Nicotinic acetylcholine receptor silent agonists

ABSTRACT

Derivatives of N,N-diethyl-N′-phenyl-piperazine, a silent agonist of the mammalian α7 nicotinic acetylcholine receptor, are provided. These silent agonists control the desensitization state of the receptor. Further provided are pharmaceutical compositions that allow the administration of the silent agonists of the disclosure to a subject animal or human in need of treatment for a pathological condition arising from such as inflammation. The novel silent agonists also may be co-administered to a patient simultaneously or consecutively with a type II positive allosteric modulator to modulate the activity of the receptor.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/241,885, entitled “DISSECTION OF N,N-DIETHYL-N-PHENYLPIPERAZINES ASNICOTINIC RECEPTOR SILENT AGONISTS” filed on Oct. 15, 2015, the entiretyof which is herein incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under contract GM57481awarded by the National Institutes of Health. The Government has certainrights in the invention.

TECHNICAL FIELD

The present disclosure is generally related to nicotinic acetylcholinereceptor silent agonists. The present disclosure is also generallyrelated to the use of the nicotinic acetylcholine receptor silentagonists in therapeutic compositions for treating inflammatorydisorders.

BACKGROUND

The homopentameric α7 nicotinic acetylcholine receptor (nAChR) is aligand-gated ion channel (Papke, R. L. (2014) Biochem. Pharmacol.)characterized by a unique form of concentration-dependent rapiddesensitization (Papke, & Papke (2002) Br. J. Pharm. 137: 49-61; Papke &Thinschmidt (1998) Neurosci. Let. 256: 163-166). The receptor belongs tothe large superfamily of ligand-gated ion channels (Papke, R. L. (2014)Biochem. Pharmacol.) that are all characterized by adisulfide-constrained “Cys-loop”, which is thought to be involved in theconformational changes linking ligand binding and ion channelactivation.

Nicotinic acetylcholine receptors (nAChRs) are validated therapeutictargets for several pathologies of the central and peripheral nervoussystem including Alzheimer's and Parkinson's diseases, addictiondisorders, schizophrenia, pain management, and inflammation-mediatedprocesses. Recent reports have provided evidence for expression of thisreceptor subtype in non-neuronal cells including lymphocytes,macrophages, and intestinal and lung endothelial and epithelial cells(Parrish et al., (2008) Mol. Med. 14: 567-574; Rosas-Ballina et al.,(2009) Mol. Med. 15: 195-202; Al-Wadei et al., (2012) Mol. Cancer Res.10: 239-249; Matteoli et al., (2014) Gut 63: 938-948), indicating anon-synaptic role for the receptor. Moreover, α7 is a key part of thecholinergic anti-inflammatory response (Wang et al., (2003) Nature 421:384-388) in which levels of pro-inflammatory cytokines are decreased(Tracey, K. J. (2007) J. Clin. Invest. 117: 289-296), making thisreceptor of great interest considering the wide range of diseases inwhich systemic inflammation is present. Further, nicotine and otheraagonists (Saeed et al., (2005) J. Exp. Med. 201: 1113-1123) have beeneffective in models of inflammation, inhibiting local leukocyterecruitment and reducing endothelial cell activation, implicating α7nAChR involvement in regulation of inflammatory processes. All thesedata makes the α7 receptor a promising drug target for the treatment ofseveral neurological disorders including inflammatory diseases andchronic pain. Notably, anti-inflammatory effects has been associatedwith desensitized, non-conducting states of the receptor (Thomsen &Mikkelsen (2012) J. Neuroimmunol. 251: 65-72; Papke et al., (2015)NeuroPharm. 91: 34-42). Thus, compounds that are able to selectivelyplace the receptor into a desensitized state rather than act as partialagonists are of considerable interest (Papke et al., (2015) NeuroPharm.91: 34-42). For the α7 receptor two distinct desensitized states havebeen identified (Williams et al., (2011) Mol. Pharmacol. 80: 1013-1032).They differ in being sensitive (D_(s)) or insensitive (D_(i)) toconversion to open states by a type II PAM. Silent agonists aredesensitizing compounds that have been identified and designed toselectively induce the D_(s) state in the α7 nAChR with extremely low orabsent partial agonist activity. The archetype compound1,4-diazabicyclo[3.2.2]nonan-4-yl(5-(3-(trifluoromethyl) phenyl)furan-2-yl) methanone (NS6740), lacking in the ability to generate an α7ion current, is an example of a silent agonist associated withanti-inflammatory activity; however, even the simple tetraethyl ammoniumcation is a silent agonist for the α7 nAChR (Papke et al., (2015)NeuroPharm. 91: 34-42; Chojnacka et al., (2013) Bioorg. Med. Chem. Lett.23: 4145-4149).

It was recently reported that the compound N,N-diethyl-N′-phenylpiperazine (diEPP) (Papke et al., (2014) J. Pharmacol. ExperimentalTherap. 350: 665-680) is also active as a silent agonist, and it has astructure that lends itself well to functionalization to explore thepotential for further enhancement and control of silent agonistactivity.

SUMMARY

The structure of the N,N-diethyl-N′-phenyl piperazine (diEPP) frameworkwas modified in three ways: different substituents on the phenyl ring,based on the identity of the functional groups and their position, thenature of the linkage between the two rings to test the essentiality ofthe N-aryl linkage, and generated monoethyl tertiary amine analogs ofdiEPP to test if a hard positive charge at the quaternary nitrogen wasrequired for silent agonism.

Briefly described, one aspect of the disclosure encompasses embodimentsof a compound having the formula I, II, III, or IV:

or a pharmaceutically acceptable salt thereof, wherein:

Y can be:

Z can be

n can be 1, 2, 3, or 4; R₁, R₂, and R₃ can each be independently ahydrogen, an alkyl group, cyano, an alkoxy group, a halogen, atrihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl, andwherein the halogen is fluorine, chlorine or bromine; R₄ can be ahydrogen or an ethyl group; and R₅ can be a hydrogen or acarboxytrifluoromethyl.

In some embodiments of this aspect of the disclosure, R₁, R₂, and R₃ caneach be independently a hydrogen, a methyl, cyano, methoxy, a halogen,trihaloalkyl, a carboxamide or hydroxyl.

In some embodiments of this aspect of the disclosure, R₁, R₂, and R₃ caneach be independently a hydrogen, a methyl, cyano, methoxy, a halogen, atrihaloalkyl, a carboxamide or hydroxyl, and R₅ is hydrogen.

In some embodiments of this aspect of the disclosure, when R₁ is amethyl or a CN, R₂ and R₃ are each hydrogen; when R₁ is a methoxy, R₂ ishydrogen R₃ is hydrogen or methoxy; when R₁ is a halogen, R₂ and R₃ areeach hydrogen; when R₁ is a trifluoromethyl, R₂ and R₃ are eachhydrogen; when R₁ is a carboxamide, pentafluorosulfanyl, R₂ and R₃ areeach hydrogen; when R₁ and R₃ are each hydrogen, R₂ is a methyl, a CN, amethoxy, a halogen, a trifluoromethyl, or a carboxamide, or OH; when R₁and R₂ are each hydrogen, R₃ is a methyl or Cl; and R₄ is an ethylgroup.

Another aspect of the disclosure encompasses embodiments of apharmaceutical composition comprising a compound having the formula I,II, III, or IV:

or a pharmaceutically acceptable salt thereof, wherein:

Y can be:

Z can be

n can be 1, 2, 3, or 4; R₁, R₂, and R₃ can each be independently ahydrogen, an alkyl group, cyano, an alkoxy group, a halogen, atrihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl, andwherein the halogen is fluorine, chlorine or bromine; R₄ can be ahydrogen or an ethyl group; and R_(s) can be a hydrogen or acarboxytrifluoromethyl; and a pharmaceutically acceptable carrier.

In some embodiments of this aspect of the disclosure, R₁, R₂, and R₃ caneach be independently a hydrogen, a methyl, cyano, methoxy, a halogen,trihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl.

In some embodiments of this aspect of the disclosure, R₁, R₂, and R₃ caneach be independently a hydrogen, a methyl, cyano, methoxy, a halogen, atrihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl, and R_(s)is hydrogen.

In some embodiments of this aspect of the disclosure, when R₁ is amethyl or a CN, R₂ and R₃ are each hydrogen; when R₁ is a methoxy, R₂ ishydrogen R₃ is hydrogen or methoxy; when R₁ is a halogen, R₂ and R₃ areeach hydrogen; when R₁ is a trifluoromethyl, R₂ and R₃ are eachhydrogen; when R₁ is a carboxamide, pentafluorosulfanyl, R₂ and R₃ areeach hydrogen; when R₁ and R₃ are each hydrogen, R₂ is a methyl, a CN, amethoxy, a halogen, a trifluoromethyl, or a carboxamide, or OH; when R₁and R₂ are each hydrogen, R₃ is a methyl or Cl; and R₄ is an ethylgroup.

In some embodiments of this aspect of the disclosure, the compositioncan be formulated to deliver to a human or animal subject in needthereof, an amount of the compound effective in modulating the activityof a nicotinic acetylcholine receptor in the recipient patient, andwherein the effective amount can delivered as a single dose or as aseries of doses.

In some embodiments of this aspect of the disclosure, the nicotinicacetylcholine receptor positive allosteric modulator (PAM) can be a typeII PAM.

In some embodiments of this aspect of the disclosure, the nicotinicacetylcholine receptor positive allosteric modulator (PAM) can be thetype II PAM1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea(PNU-120596).

Yet another aspect of the disclosure encompasses a method of modulatingthe activity of a nicotinic acetylcholine receptor in an animal or humansubject by administering to said subject effective doses of a silentagonist of the nicotinic acetylcholine receptor and a nicotinicacetylcholine receptor positive allosteric modulator (PAM).

In some embodiments of this aspect of the disclosure, the silent agonistof the nicotinic acetylcholine receptor and the nicotinic acetylcholinereceptor positive allosteric modulator (PAM) can be administered to thesubject simultaneously or as consecutive doses.

In some embodiments of this aspect of the disclosure, the silent agonistof the nicotinic acetylcholine receptor is a compound having the formulaI, II, III, or IV:

or a pharmaceutically acceptable salt thereof, wherein:

Y is:

Z is

n can be 1, 2, 3, or 4; R₁, R₂, and R₃ can each be independently ahydrogen, an alkyl group, cyano, an alkoxy group, a halogen, atrihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl, andwherein the halogen is fluorine, chlorine or bromine; R₄ is a hydrogenor an ethyl group; and R_(s) is a hydrogen or a carboxytrifluoromethyl.

In some embodiments of this aspect of the disclosure, the nicotinicacetylcholine receptor positive allosteric modulator (PAM) can be a typeII PAM.

In some embodiments of this aspect of the disclosure, the nicotinicacetylcholine receptor positive allosteric modulator (PAM) can be thetype II PAM1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea(PNU-120596).

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be more readilyappreciated upon review of the detailed description of its variousembodiments, described below, when taken in conjunction with theaccompanying drawings.

FIG. 1 schematically illustrates a synthetic approach to compounds 1 and2. Reagents and reaction conditions: (a) 2 equivalents of K₂CO₃ (X═I) orK₂PO₄ (X═Br), 0.2 equivalent of proline, 0.1 equivalents of CuI, DMSO,90-100° C., 14 h to 112.5 h; (b) 7 equivalents of Etl, THF, 80-90° C.,17 h to 68.5 h; (c) 2 equivalents of acetaldoxime, 0.05 equivalents ofPd(PPH₃)₄, EtOH, reflux, 63 h to 87 h.

FIG. 2 schematically illustrates a synthetic approach to compounds 6 and8. Reagents and reaction conditions: (a) 1.2 equivalents of1-ethyl-4-piperidone, 1 equivalent of aniline, 1.2 equivalents ofNaBH₃CN, AcOH to pH 6-7, dry MeOH, RT to reflux, 7d; (b) 3 equivalentsof TFAA, 4 equivalents of TEA, dry CH₂Cl₂, 0° C., 5 h; (c) 7 equivalentsof Etl, copper metal, dry THF, 90° C., 22 h; (d) 12 equivalents ofK₂CO₃, MeOH/H₂O 7:1, RT, 1 h; (e) 1 equivalent of 1-ethyl-4-piperidone,1.2 equivalents of diethylbenzylphosphonate, 1.5 equivalents of NaH, 0.2equivalents of 15-crown-5, dry THF, 0° C. to RT, 4d; 7 equivalents ofEtl, copper metal, EtOH, 90° C., 2d.

FIG. 3A illustrates the structures of six test compounds that wereclassified as partial agonists.

FIG. 3B illustrates the responses of oocytes expressing α7 to theapplication of the compounds at the probe concentration of 30 μM. Ineach trace, the black line is the average of the normalized response ofat least four cells, and the shaded areas are the range of the standarderror of the mean for each point in the averaged traces. The data wereobtained at 50 Hz and the traces are each 55 secs in duration (2750points in each trace). Two control responses to 60 μM ACh were firstobtained from each cell for purposes of normalization. The drug-evokedresponses were scaled in amplitude to the average of the two ACh-evokedcontrol responses. A representative set of averaged ACh responses areshown on the left to provide scale. Those shown are the ACh responsesobtained prior to the application of 30 μM p-cyano. Note that thedrug-evoked responses vary in amplitude and also in regard to the ratioof peak currents to net charge (area) calculated relative to the peakand net charge of the ACh controls (Table 2). This ratio is indicativeof the drug's potency, i.e. the relationship between the testconcentration and EC50 21.

FIG. 3C illustrates the responses of oocytes expressing α7 to theapplication of the compounds at the probe concentration of 30 μMco-applied with 10 μM1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea(PNU-120596). Data were normalized and averaged as described for FIG.3B. For comparison and scale, shown on the left is the averaged AChresponse obtained prior to the application of 30 μM p-cyano plus 10 μMPNU-120596. All of the traces are reduced 40-fold relative to those inFIG. 3B.

FIGS. 4A-4C illustrate the activity of diEPP compounds and analogs withthe α7 nAChR. The left vertical axis refers to the net charge responseof compounds when compounds (30 μM) were co-applied with 10 μMPNU-120596, relative to ACh controls. Experimental values are theaverage of at least 4 independent measurements and the error barsreflect the calculated standard deviation of the mean. In all casesthese compounds showed less than 10% of the control ACh response whenthey were applied alone to the receptor. For reference, all figuresinclude the reference response of diEPP.

FIG. 4A is a graph illustrating the data for the meta-substitutedcompounds 2.

FIG. 4B is a graph illustrating the data for the para-substitutedcompounds 2.

FIG. 4C is a graph illustrating the data for selected compounds 1 (leftside) and compounds 6 and 8 (right side).

FIG. 5 illustrates that silent agonists have low efficacy for activatingthe α7 ion channel when applied alone but induce desensitized statesthat can be converted to active states with a positive allostericmodulator like PNU-120596.

FIG. 6 illustrates that the silent agonist NS6740 induces a desensitizedstate that can be converted to an active state with a positiveallosteric modulator like PNU-120596.

FIG. 7 illustrates the minimal pharmacophore for silent agonism of theα7 nicotinic acetylcholine receptor (nAChR).

FIG. 8 illustrates the structure of the type II PAM of α7 nAChR,PNU-120596.

FIG. 9 illustrates representative traces of the hα7 nAChR response tothe application of a silent agonist and PNU-120596.

FIG. 10 is a graph illustrating the net-charge responses of oocytesexpressing human α7 receptors for diEPP.

FIG. 11 is a graph illustrating the net-charge responses of oocytesexpressing human α7 receptors for diEPP derivatives found to silentagonists. diEPP derivatives were tested in one shot experiments at 30μM, with 60 μM ACh as control.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise. In this disclosure, “comprises,”“comprising,” “containing” and “having” and the like can have themeaning ascribed to them in U.S. patent law and can mean “includes,”“including,” and the like; “consisting essentially of” or “consistsessentially” or the like, when applied to methods and compositionsencompassed by the present disclosure refers to compositions like thosedisclosed herein, but which may contain additional structural groups,composition components or method steps (or analogs or derivativesthereof as discussed above). Such additional structural groups,composition components or method steps, etc., however, do not materiallyaffect the basic and novel characteristic(s) of the compositions ormethods, compared to those of the corresponding compositions or methodsdisclosed herein.

Prior to describing the various embodiments, the following definitionsare provided and should be used unless otherwise indicated.

Abbreviations

nAChR, nicotinic acetylcholine receptor; diEPP,N,N-diethyl-N′-phenyl-piperazine, PAM, positive allosteric modulator,PAM; i.p., intraperitoneal

Definitions

The term “nicotinic acetylcholine receptors (nAChRs)” as used hereinrefers to pentameric integral membrane proteins that are members of afamily of ligand-gated ion channel receptors, which include theGABA_(A), glycine, and serotonin 5HT3_(A) and _(B) receptors. The nAChRsmediate “fast” synaptic transmission on a millisecond time frame,rapidly changing the membrane potential. Each of the five constituentreceptor polypeptide subunits share a common motif that includes a largeextracellular N-terminal hydrophilic domain, three transmembranehydrophobic domains (termed M1-M3), an intracellular loop of variablesize that contains consensus sequences of amino acids for enzymaticphosphorylation, and a C-terminal M4 transmembrane hydrophobic domain;the M2 transmembrane domains of each of the five receptor polypeptidesubunits are aligned to create a potential channel, whose opening isgated by acetylcholine. These receptors are assembled from an extensivefamily of subunits. In vertebrates, the 17 nAChR subunits (α1-α10,β1-β4, γ, δ, and ε) can assemble into a variety of pharmacologicallydistinct receptor subtypes. There are muscle-type nAChRs and neuronalnAChRs. There is considerable diversity among the sub-family of neuronalnAChRs.

The term “alkoxy” as used herein refers to a linear or branchedoxy-containing radical having an alkyl portion of one to about tencarbon atoms, such as a methoxy radical, which may be substituted. Inaspects of the disclosure an alkoxy radical may comprise about 1-10,1-8, 1-6 or 1-3 carbon atoms. In embodiments of the disclosure, analkoxy radical comprises about 1-6 carbon atoms and includes a C₁-C₆alkyl-O-radical wherein C₁-C₆ alkyl has the meaning set out herein.Examples of alkoxy radicals include without limitation methoxy, ethoxy,propoxy, butoxy, isopropoxy and tert-butoxy alkyls. An “alkoxy” radicalmay, optionally be substituted with one or more substitutents disclosedherein including alkyl atoms to provide “alkylalkoxy” radicals; haloatoms, such as fluoro, chloro or bromo, to provide “haloalkoxy” radicals(e.g. fluoromethoxy, chloromethoxy, trifluoromethoxy, difluoromethoxy,trifluoroethoxy, fluoroethoxy, tetrafluoroethoxy, pentafluoroethoxy, andfluoropropox) and “haloalkoxyalkyl” radicals (e.g. fluoromethoxymethyl,chloromethoxyethyl, trifluoromethoxymethyl, difluoromethoxyethyl, andtrifluoroethoxymethyl).

The term “alkyl”, either alone or within other terms such as “thioalkyl”and “arylalkyl”, as used herein refers to a monovalent, saturatedhydrocarbon radical which may be a straight chain (i.e. linear) or abranched chain. An alkyl radical for use in the present disclosuregenerally comprises from about 1 to 20 carbon atoms, particularly fromabout 1 to 10, 1 to 8 or 1 to 7, more particularly about 1 to 6 carbonatoms, or 3 to 6. Illustrative alkyl radicals include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, isopentyl,amyl, sec-butyl, tert-butyl, tert-pentyl, n-heptyl, n-octyl, n-nonyl,n-decyl, undecyl, n-dodecyl, n-tetradecyl, pentadecyl, n-hexadecyl,heptadecyl, n-octadecyl, nonadecyl, eicosyl, dosyl, n-tetracosyl, andthe like, along with branched variations thereof. In certain aspects ofthe disclosure an alkyl radical is a C₁-C₆ lower alkyl comprising orselected from the group consisting of methyl, ethyl, n-propyl, n-butyl,n-pentyl, n-hexyl, isopropyl, isobutyl, isopentyl, amyl, tributyl,sec-butyl, tert-butyl, tert-pentyl, and n-hexyl. An alkyl radical may beoptionally substituted with substituents as defined herein at positionsthat do not significantly interfere with the preparation of compounds ofthe disclosure and do not significantly reduce the efficacy of thecompounds. In certain aspects of the disclosure, an alkyl radical issubstituted with one to five substituents including halo, lower alkoxy,lower aliphatic, a substituted lower aliphatic, hydroxy, cyano, nitro,thio, amino, keto, aldehyde, ester, amide, substituted amino, carboxyl,sulfonyl, sulfuryl, sulfenyl, sulfate, sulfoxide, substituted carboxyl,halogenated lower alkyl (e.g. CF₃), halogenated lower alkoxy,hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy, loweralkylcarbonylamino, cycloaliphatic, substituted cycloaliphatic, or aryl(e.g., phenylmethyl benzyl)), heteroaryl (e.g., pyridyl), andheterocyclic (e.g., piperidinyl, morpholinyl). Substituents on an alkylgroup may themselves be substituted.

The term “cycloalkyl” as used herein refers to radicals having fromabout 3 to 8, or 3 to 6 carbon atoms and containing one or two suchrings that may be attached in a pendant manner or may be fused. Examplesof cycloalkyl groups include single ring structures such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,cyclononyl, cyclodecyl, cyclododecyl, and the like.

The aryl group can be optionally substituted (a “substituted aryl”) withone or more aryl group substituents, which can be the same or different,wherein “aryl group substituent” includes alkyl, substituted alkyl,aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl,aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl,aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino,carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio,alkylene, and —NR′R″, wherein R′ and R″ can each be independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl.

The term “carbonyl” as used herein refers to a carbon radical having twoof the four covalent bonds shared with an oxygen atom. Carbonyl- Theterm “carbonyl” as used herein refers to the —(C=O)— group.

The term “carboxamide” as used herein refers to the group —CONH—.

The Term “carboxyl” as used herein refers to the —COOH group.

The terms “co-administration” or “co-administered” as used herein referto the administration of at least two compounds or agent(s) or therapiesto a subject. In some embodiments, the co-administration of two or moreagents/therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy in thisaspect, each component may be administered separately, but sufficientlyclose in time to provide the desired effect, in particular a beneficial,additive, or synergistic effect. Those of skill in the art understandthat the formulations and/or routes of administration of the variousagents/therapies used may vary. The appropriate dosage forco-administration can be readily determined by one skilled in the art.In some embodiments, when agents/therapies are co-administered, therespective agents/therapies are administered at lower dosages thanappropriate for their administration alone. Thus, co-administration isespecially desirable in embodiments where the co-administration of theagents/therapies lowers the requisite dosage of a known potentiallyharmful (e.g., toxic) agent(s).

The term “composition” as used herein refers to a product comprising thespecified ingredients in the specified amounts, as well as any productwhich results, directly or indirectly, from combination of the specifiedingredients in the specified amounts. Such a term in relation to apharmaceutical composition is intended to encompass a product comprisingthe active ingredient(s), and the inert ingredient(s) that make up thecarrier, as well as any product which results, directly or indirectly,from combination, complexation, or aggregation of any two or more of theingredients, or from dissociation of one or more of the ingredients, orfrom other types of reactions or interactions of one or more of theingredients. Accordingly, the pharmaceutical compositions of the presentdisclosure encompass any composition made by admixing a compound of thepresent disclosure and a pharmaceutically acceptable carrier.

When a compound of the present disclosure is used contemporaneously withone or more other drugs, a pharmaceutical composition containing suchother drugs in addition to the compound of the present disclosure iscontemplated. Accordingly, the pharmaceutical compositions of thepresent disclosure include those that also contain one or more otheractive ingredients, in addition to a compound of the present disclosure.The weight ratio of the compound of the present disclosure to the secondactive ingredient may be varied and will depend upon the effective doseof each ingredient. Generally, an effective dose of each will be used.Thus, for example, but not intended to be limiting, when a compound ofthe present disclosure is combined with another agent, the weight ratioof the compound of the present disclosure to the other agent willgenerally range from about 1000:1 to about 1:1000, preferably about200:1 to about 1:200. Combinations of a compound of the presentdisclosure and other active ingredients will generally also be withinthe aforementioned range, but in each case, an effective dose of eachactive ingredient should be used. In such combinations the compound ofthe present disclosure and other active agents may be administeredseparately or in conjunction. In addition, the administration of oneelement may be prior to, concurrent to, or subsequent to theadministration of other agent(s).

A therapeutic composition of the disclosure may comprise a carrier, suchas one or more of a polymer, carbohydrate, peptide or derivativethereof, which may be directly or indirectly covalently attached to thecompound. A carrier may be substituted with substituents describedherein including without limitation one or more alkyl, amino, nitro,halogen, thiol, thioalkyl, sulfate, sulfonyl, sulfinyl, sulfoxide,hydroxyl groups. In aspects of the disclosure the carrier is an aminoacid including alanine, glycine, praline, methionine, serine, threonine,asparagine, alanyl-alanyl, prolyl-methionyl, or glycyl-glycyl. A carriercan also include a molecule that targets a compound of the disclosure toa particular tissue or organ.

A compound of the disclosure may be in the form of a prodrug that isconverted in vivo to an active compound.

Compounds of the disclosure can be prepared using reactions and methodsgenerally known to the person of ordinary skill in the art, havingregard to that knowledge and the disclosure of this applicationincluding the Examples. The reactions are performed in solventappropriate to the reagents and materials used and suitable for thereactions being effected. It will be understood by those skilled in theart of organic synthesis that the functionality present on the compoundsshould be consistent with the proposed reaction steps. This willsometimes require modification of the order of the synthetic steps orselection of one particular process scheme over another in order toobtain a desired compound of the disclosure. It will also be recognizedthat another major consideration in the development of a synthetic routeis the selection of the protecting group used for protection of thereactive functional groups present in the compounds described in thisdisclosure. An authoritative account describing the many alternatives tothe skilled artisan is Greene and Wuts (Protective Groups In OrganicSynthesis, Wiley and Sons, 1991).

Compounds of the disclosure which are acidic in nature are capable offorming base salts with various pharmacologically acceptable cations.These salts may be prepared by conventional techniques by treating thecorresponding acidic compounds with an aqueous solution containing thedesired pharmacologically acceptable cations and then evaporating theresulting solution to dryness, preferably under reduced pressure.Alternatively, they may be prepared by mixing lower alkanolic solutionsof the acidic compounds and the desired alkali metal alkoxide togetherand then evaporating the resulting solution to dryness in the samemanner as before. In either case, stoichiometric quantities of reagentsare typically employed to ensure completeness of reaction and maximumproduct yields.

The compounds of the disclosure which are basic in nature can form awide variety of different salts with various inorganic and organicacids. In practice is it desirable to first isolate a compound of thedisclosure from a reaction mixture as a pharmaceutically unacceptablesalt and then convert the latter to the free base compound by treatmentwith an alkaline reagent and subsequently convert the free base to apharmaceutically acceptable acid addition salt. The acid addition saltsof the base compounds of the disclosure are readily prepared by treatingthe base compound with a substantially equivalent amount of the chosenmineral or inorganic or organic acid in an aqueous solvent medium or ina suitable organic solvent such as methanol or ethanol. Upon carefulevaporation of the solvent, the desired solid salt is obtained.

A composition of the disclosure may be sterilized by, for example,filtration through a bacteria retaining filter, addition of sterilizingagents to the composition, irradiation of the composition, or heatingthe composition. Alternatively, the compounds or compositions of thepresent disclosure may be provided as sterile solid preparations e.g.lyophilized powder, which are readily dissolved in sterile solventimmediately prior to use.

A compound of the disclosure includes crystalline forms which may existas polymorphs. Solvates of the compounds formed with water or commonorganic solvents are also intended to be encompassed within the term. Inaddition, hydrate forms of the compounds and their salts are encompassedwithin this disclosure. Further prodrugs of compounds of the disclosureare encompassed within the term.

The term “cyano” as used herein refers to a carbon radical having threeof four covalent bonds shared by a nitrogen atom, in particular —CN. Acyano group may be substituted with substituents described herein.

A compound of the disclosure includes derivatives. As used herein theterm “derivative” of a compound of the disclosure refers to a chemicallymodified compound wherein the chemical modification takes place eitherat a functional group or ring of the compound. Non-limiting examples ofderivatives of compounds of the disclosure may include N-acetyl,N-methyl, N-hydroxy groups at any of the available nitrogens in thecompound.

A compound of the disclosure can contain one or more asymmetric centersand may give rise to enantiomers, diasteriomers, and otherstereoisomeric forms which may be defined in terms of absolutestereochemistry as (R)- or (S)-. Thus, compounds of the disclosureinclude all possible diasteriomers and enantiomers as well as theirracemic and optically pure forms. Optically active (R)- and (S)-isomersmay be prepared using chiral synthons or chiral reagents, or resolvedusing conventional techniques. When a compound of the disclosurecontains centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and A geometricisomers. All tautomeric forms are also included within the scope of acompound of the disclosure.

The terms “effective amount,” “therapeutically-effective amount,” and“therapeutically effective dose” as used herein refer to the amount of acompound, material, or composition comprising a compound or compositionof the present disclosure, and which is effective for producing adesired therapeutic effect, biological or medicinal response in a tissuesystem, animal or human that is being sought by a researcher,veterinarian, medical doctor or other clinician, which includesalleviation of the symptoms of the disease or disorder being treated ora reduction in a side-effect due to an administered pharmaceuticalagent.

The term “excipient” as used herein refers to an inert substance addedto a pharmaceutical composition to further facilitate administration ofa compound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

The term “halo” as used herein refers to a halogen such as fluorine,chlorine, bromine or iodine atoms.

The term “hydroxyl” as used herein refers to the —OH group.

The term “modulate” refers to the activity of a composition to affect(e.g., to promote or retard) an aspect of cellular function, including,but not limited to, cell growth, proliferation, apoptosis, and the like.

The term “pharmaceutically acceptable carrier” as used herein refers toa diluent, adjuvant, excipient, or vehicle with which a probe of thedisclosure is administered and which is approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. Such pharmaceutical carriers can be liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. The pharmaceutical carriers can be saline, gumacacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,and the like. When administered to a patient, the probe andpharmaceutically acceptable carriers can be sterile. Water is a usefulcarrier when the probe is administered intravenously. Saline solutionsand aqueous dextrose and glycerol solutions can also be employed asliquid carriers, particularly for injectable solutions. Suitablepharmaceutical carriers also include excipients such as glucose,lactose, sucrose, glycerol monostearate, sodium chloride, glycerol,propylene, glycol, water, ethanol and the like. The presentcompositions, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. The present compositionsadvantageously may take the form of solutions, emulsion,sustained-release formulations, or any other form suitable for use.

The compounds of the disclosure may also include “pharmaceuticallyacceptable salt(s)”. By pharmaceutically acceptable salts is meant thosesalts which are suitable for use in contact with the tissues of asubject or patient without undue toxicity, irritation, allergic responseand the like, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are described for example, in Berge,et al., J. Pharmaceut. Sci., 1977, 66: 1. Suitable salts include saltsthat may be formed where acidic protons in the compounds are capable ofreacting with inorganic or organic bases. Suitable inorganic saltsinclude those formed with alkali metals, e.g. sodium and potassium,magnesium, calcium, and aluminum. Suitable organic salts include thoseformed with organic bases such as the amine bases, e.g. ethanolamine,diethanolamine, triethanolamine, trimethamine, N-methylglucamine, andthe like. Suitable salts also include acid addition salts formed withinorganic acids (e.g. hydrochloric and hydrobromic acids) and organicacids (e.g. acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). When there are two acidic groups present, a pharmaceuticallyacceptable salt may be a mono-acid-mono-salt or a di-salt; and similarlywhere there are more than two acidic groups present, some or all of suchgroups can be salified.

The term “pharmaceutically acceptable” as used herein refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The term “substituted alkyl” includes alkyl groups, as defined herein,in which one or more atoms or functional groups of the alkyl group arereplaced with another atom or functional group, including for example,alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl,hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

The term “substituted aryl” as used herein includes an aromatic ring, orfused aromatic ring system consisting of no more than three fused ringsat least one of which is aromatic, and where at least one of thehydrogen atoms on a ring carbon has been replaced by a halogen, anamino, a hydroxy, a nitro, a thio, an alkyl, a ketone, an aldehyde, anester, an amide, a lower aliphatic, a substituted lower aliphatic, or aring (aryl, substituted aryl, cycloaliphatic, or substitutedcycloaliphatic). Examples of such include, but are not limited to,hydroxyphenyl, chlorphenyl and the like.

In the event that embodiments of the disclosed compounds in thecomposition or pharmaceutical composition form salts, these salts arewithin the scope of the present disclosure. Reference to a compound usedin the composition or pharmaceutical composition of any of the formulasherein is understood to include reference to salts thereof, unlessotherwise indicated. The term “salt(s)”, as employed herein, denotesacidic and/or basic salts formed with inorganic and/or organic acids andbases. In addition, when a compound contains both a basic moiety and anacidic moiety, zwitterions (“inner salts”) may be formed and areincluded within the term “salt(s)” as used herein. Pharmaceuticallyacceptable (e.g., non-toxic, physiologically acceptable) salts arepreferred, although other salts are also useful, e.g., in isolation orpurification steps which may be employed during preparation. Salts ofthe compounds of a compound may be formed, for example, by reacting thecompound with an amount of acid or base, such as an equivalent amount,in a medium such as one in which the salt precipitates or in an aqueousmedium followed by lyophilization.

Embodiments of the compounds of the composition or pharmaceuticalcomposition of the present disclosure that contain a basic moiety mayform salts with a variety of organic and inorganic acids. Exemplary acidaddition salts include acetates (such as those formed with acetic acidor trihaloacetic acid, for example, trifluoroacetic acid), adipates,alginates, ascorbates, aspartates, benzoates, benzenesulfonates,bisulfates, borates, butyrates, citrates, camphorates,camphorsulfonates, cyclopentanepropionates, digluconates,dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates,glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides(formed with hydrochloric acid), hydrobromides (formed with hydrogenbromide), hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates(formed with maleic acid), methanesulfonates (formed withmethanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates,oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates,picrates, pivalates, propionates, salicylates, succinates, sulfates(such as those formed with sulfuric acid), sulfonates (such as thosementioned herein), tartrates, thiocyanates, toluenesulfonates such astosylates, undecanoates, and the like.

Embodiments of the compounds of the composition or pharmaceuticalcomposition of the present disclosure that contain an acidic moiety mayform salts with a variety of organic and inorganic bases. Exemplarybasic salts include ammonium salts, alkali metal salts such as sodium,lithium, and potassium salts, alkaline earth metal salts such as calciumand magnesium salts, salts with organic bases (for example, organicamines) such as benzathines, dicyclohexylamines, hydrabamines (formedwith N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines,N-methyl-D-glucamides, t-butyl amines, and salts with amino acids suchas arginine, lysine, and the like.

Basic nitrogen-containing groups may be quaternized with agents such aslower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides,bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl,dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl,myristyl and stearyl chlorides, bromides and iodides), aralkyl halides(e.g., benzyl and phenethyl bromides), and others.

Solvates of the compounds of the composition or pharmaceuticalcomposition of the present disclosure are also contemplated herein.

To the extent that the disclosed the compounds of the composition orpharmaceutical composition of the present disclosure, and salts thereof,may exist in their tautomeric form, all such tautomeric forms arecontemplated herein as part of the present disclosure.

All stereoisomers of the compounds of the composition or pharmaceuticalcomposition of the present disclosure, such as those that may exist dueto asymmetric carbons on the various substituents, includingenantiomeric forms (which may exist even in the absence of asymmetriccarbons) and diastereomeric forms, are contemplated within the scope ofthis disclosure. Individual stereoisomers of the compounds of thedisclosure may, for example, be substantially free of other isomers, ormay be admixed, for example, as racemates or with all other, or otherselected, stereoisomers. The stereogenic centers of the compounds of thepresent disclosure can have the S or R configuration as defined by theIUPAC 1974 Recommendations.

The term “prodrug” refers to an inactive precursor of the compounds ofthe composition or pharmaceutical composition of the present disclosurethat is converted into a biologically active form in vivo. Prodrugs areoften useful because, in some situations, they may be easier toadminister than the parent compound. They may, for instance, bebioavailable by oral administration whereas the parent compound is not.The prodrug may also have improved solubility in pharmaceuticalcompositions over the parent drug. A prodrug may be converted into theparent drug by various mechanisms, including enzymatic processes andmetabolic hydrolysis. Harper, N.J. (1962). Drug Latentiation in Jucker,ed. Progress in Drug Research, 4:221-294; Morozowich et al. (1977).Application of Physical Organic Principles to Prodrug Design in E. B.Roche ed. Design of Biopharmaceutical Properties through Prodrugs andAnalogs, APhA; Acad. Pharm. Sci.; E. B. Roche, ed. (1977). BioreversibleCarriers in Drug in Drug Design, Theory and Application, APhA; H.Bundgaard, ed. (1985) Design of Prodrugs, Elsevier; Wang et al. (1999)Prodrug approaches to the improved delivery of peptide drug, Curr.Pharm. Design. 5(4):265-287; Pauletti et al. (1997). Improvement inpeptide bioavailability: Peptidomimetics and Prodrug Strategies, Adv.Drug. Delivery Rev. 27: 235-256; Mizen et al. (1998). The Use of Estersas Prodrugs for Oral Delivery of β-Lactam antibiotics, Pharm. Biotech.11:345-365; Gaignault et al. (1996). Designing Prodrugs andBioprecursors I. Carrier Prodrugs, Pract. Med. Chem. 671-696; M.Asgharnejad (2000). Improving Oral Drug Transport Via Prodrugs, in G. L.Amidon, P. I. Lee and E. M. Topp, Eds., Transport Processes inPharmaceutical Systems, Marcell Dekker, p. 185-218; Balant et al. (1990)Prodrugs for the improvement of drug absorption via different routes ofadministration, Eur. J. Drug Metab. Pharmacokinet., 15(2): 143-53;Balimane and Sinko (1999). Involvement of multiple transporters in theoral absorption of nucleoside analogues, Adv. Drug Delivery Rev.,39(1-3):183-209; Browne (1997). Fosphenytoin (Cerebyx), Clin.Neuropharmacol. 20(1): 1-12; Bundgaard (1979). Bioreversiblederivatization of drugs-principle and applicability to improve thetherapeutic effects of drugs, Arch. Pharm. Chemi. 86(1): 1-39; H.Bundgaard, ed. (1985) Design of Prodrugs, New York: Elsevier; Fleisheret al. (1996). Improved oral drug delivery: solubility limitationsovercome by the use of prodrugs, Adv. Drug Delivery Rev. 19(2): 115-130;Fleisher et al. (1985). Design of prodrugs for improved gastrointestinalabsorption by intestinal enzyme targeting, Methods Enzymol. 112: 360-81;Farquhar D, et al. (1983). Biologically Reversible Phosphate-ProtectiveGroups, J. Pharm. Sci., 72(3): 324-325; Han, H. K. et al. (2000).Targeted prodrug design to optimize drug delivery, AAPS PharmSci., 2(1):E6; Sadzuka Y. (2000). Effective prodrug liposome and conversion toactive metabolite, Curr. Drug Metab., 1(1):31-48; D. M. Lambert (2000)Rationale and applications of lipids as prodrug carriers, Eur. J. Pharm.Sci., 11 Suppl 2:S15-27; Wang, W. et al. (1999) Prodrug approaches tothe improved delivery of peptide drugs. Curr. Pharm. Des., 5(4):265-87.

The term “administration” refers to introducing an agent of the presentdisclosure into a host. One preferred route of administration of theagents is oral administration. Another preferred route is intravenousadministration. However, any route of administration, such as topical,subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal,nasal, introduction into the cerebrospinal fluid, or instillation intobody compartments can be used.

The terms “treatment”, “treating”, and “treat” as used herein refer toacting upon a disease, disorder, or condition with composition orpharmaceutical composition of the present disclosure to reduce orameliorate the pharmacologic and/or physiologic effects of the disease,disorder, or condition and/or its symptoms. “Treatment,” as used herein,covers any treatment of a disease in a host (e.g., a mammal, typically ahuman or non-human animal of veterinary interest), and includes: (a)reducing the risk of occurrence of the disease in a subject determinedto be predisposed to the disease but not yet diagnosed as infected withthe disease, (b) impeding the development of the disease, and (c)relieving the disease, i.e., causing regression of the disease and/orrelieving one or more disease symptoms. “Treatment” is also meant toencompass delivery of the composition or pharmaceutical composition ofthe present disclosure to provide a pharmacologic effect, even in theabsence of a disease or condition. For example, “treatment” encompassesdelivery of the composition or pharmaceutical composition of the presentdisclosure that provides for enhanced or desirable effects in thesubject (e.g., reduction of disease symptoms, etc.).

The terms “host,” “subject,” “patient,” or “organism” as used herein,includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, andhorses). Typical hosts to which compounds of the present disclosure maybe administered will be mammals, particularly primates, especiallyhumans. For veterinary applications, a wide variety of subjects will besuitable, e.g., livestock such as cattle, sheep, goats, cows, swine, andthe like; poultry such as chickens, ducks, geese, turkeys, and the like;and domesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects, including rodents (e.g., mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like. The term“living host” refers to a host noted above or another organism that isalive. The term “living host” refers to the entire host or organism andnot just a part excised (e.g., a liver or other organ) from the livinghost.

The term “agonist” as used herein refers to a compound or molecule,including but not limited to, peptides, oligopeptides, and smallmolecules, variants and derivatives thereof that may interact with areceptor of a cell, thereby inducing an increase in a biochemical orphysiological activity attributable to the receptor. The agonist may be,but is not limited to, a natural ligand effector of the receptor, ananalog or a mimetic and the like thereof.

The term “antagonist” as used herein refers to a compound or molecule,including but not limited to, peptides, oligopeptides, and smallmolecules, variants and derivatives thereof that may interact with areceptor of a cell, thereby inducing a decrease in a biochemical orphysiological activity attributable to the receptor. The agonist may be,but is not limited to, a natural inhibitor of the receptor, an analog ora mimetic and the like thereof.

The terms “subject” and “subject animal or human” as used herein refersto any animal, including a human, to which a composition according tothe disclosure may be delivered or administered.

Description

The present disclosure encompasses embodiments of derivatives ofN,N-diethyl-N′-phenyl-piperazine (diEPP), the silent agonist of themammalian α7 nicotinic acetylcholine receptor. These silent agonist canmodulate the desensitization states of the nAChR, thus modulating theactivity of the receptor.

In particular, the disclosure further encompasses embodiments ofpharmaceutical compositions that allow the administration of the silentagonists of the disclosure to a subject animal or human in need oftreatment for a pathological condition arising from such as inflammationof the central or peripheral nervous system. The silent agonist may beco-administered either simultaneously or consecutively with a positiveallosteric modulators (PAMs). The disclosure further encompassesembodiments of methods of treating a disorder of the central orperipheral nervous system by administering to a patient in need thereofa therapeutically effective amount of the silent agonist, alone or inconjunction with a PAM.

The α7 nicotinic acetylcholine receptor is a target for control ofinflammation-related phenomena via compounds that are able toselectively induce desensitized states of the receptor. Compounds thatselectively desensitize, without facilitating significant channelactivation, are termed “silent agonists” because they can be detected byco-application with type II positive allosteric modulators (PAMs). Oneexample is N,N-diethyl-N′-phenyl-piperazine (diEPP) (Papke et al.,(2014) J. Pharmacol. Experimental Therap. 350: 665-680). Ullmann-typearyl amination was used to a synthesized panel of compounds related todiEPP by substitutions at the aryl ring, the linkage between thepiperazine and phenyl rings, and the nature of substituents at thequarternary nitrogen of the piperazine ring. Two-electrode voltageclamping of the human α7 nAChR expressed in Xenopus oocytes revealedthat it was possible to tune the behavior of compounds to show enhanceddesensitization without corresponding partial agonist activity such thattrifluoromethyl and carboxamide aryl substituents showed 33 to 46-foldlarger PAM-dependent net-charge responses, indicating selectivepartitioning of the ligand-receptor complexes into the desensitizedstate.

Nicotinic acetylcholine receptors (nAChRs) belong to the fourtransmembrane domain superfamily of neurotransmitter-gated ion channelsand are composed of pentameric combinations, with a high degree ofcomplexity conferred by 10 different alpha (α1-α10) and non-alpha β1-β4,γ, δ, ε) subunits. The homomeric α7 nAChR is characterized by: greatabundancy in the CNS regions (cortex, hippocampus and auditory cortex);a unique form of concentration-dependent rapid desensitization, highpermeability to calcium ions and very low probability of channelopening; expression on non-neuronal cells, such as lymphocytes,macrophages, intestinal and lung endothelial and epithelial cells,adipocytes; multiple allosteric sites; different conformational states:a resting closed one with no ion flow through it in absence of anagonist; a very short-lasting cation-permeable open state; adesensitized state when the agonist is bound but the receptor is closedand no activation can occur.

In particular, depending on the agonist nature, residual inhibition ordesensitization can be present. Two different desensitized states,namely Ds and Di (sensitive or insensitive to conversion to open statesmediated by a type II positive allosteric modulator) are possible andthey might involve different intracellular signaling pathways.

α7 nAChR and inflammation: Targeting α7 nAChRs represents a viable andpromising therapeutic strategy for a broad array of intractable diseasesand conditions with inflammatory components. According to preclinicalstudies, the α7 nAChR is involved in inflammatory processes throughmodulation of pro-inflammatory cytokines. Indeed, the “cholinergicanti-inflammatory pathway” modulates the immune system through α7receptors expressed on macrophages and immune cells, down-regulatingproinflammatory cytokine synthesis and preventing tissue damage.

Moreover, α7 nAChR modulates inflammatory genes expression in humanadipocytes and its expression levels are significantly decreased inobese subjects. Thus, the α7 nAChR represents a useful target to reducethe low-grade chronic inflammation associated with severe human obesityand the subsequent onset of insulin resistance and Type II diabetes.

The development of new drugs with increased efficacy and safety is ofgreat interest since currently-used agents suffer from major adverseeffects and/or incomplete pain relief. The development of new α7anti-inflammatory drugs has been traditionally focused on selectiveactivators. However the best α7 compounds to address treatment ofchronic pain and inflammation may not belong to that category. To thisend, the α7 receptor activation was investigated by means of silentagonists, which work by involving non-ion conducting states of thereceptor.

A new class of compounds named silent agonists has now been discovered(Chojnacka et al., (2013) Bioorg. Med. Chem. Lett. 23: 4145-4149). An α7silent agonist (FIG. 10) is defined as a molecule that: binds at theorthosteric site of the receptor, but it is not able to activate, oronly very weakly, the receptor channel opening when used alone, itinhibits an α7 response to Ach, and induces preferentiallynon-conducting α7 receptor states (Ds or Di), and therefore it appearslike an agonist when co-applied with a type II PAM. The type II PAMPNU-120596(1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea)destabilizes a form of desensitization unique to the α7 receptorsubtype, 1 thereby promoting protracted bursts of channel opening,overcoming the very low channel open probability of α7.

Even in absence of the PAM, receptors in the Ds and/or Di states arepotential mediators of signal transduction. The idea of α7 signaltrasduction independent to ion channel currents is supported by resultscollected on the silent agonist NS-6740, studied as a modulator of theinflammatory function of microglia. This compound was more efficaciousthan some agonists (including choline) in suppressing LPS-stimulatedsecretion of TNF-α in rat cultured microglia; in in vivo mouse painmodels showed antinoceptive activity in formalin- and aceticacid-induced nociceptive conditions, and in pain due to the chronicconstrictive nerve injury (significantly dose- and time-dependent).

Novel potential silent agonist derivatives: diEPP derivatives: Thesilent agonist diEPP (1,1-diethyl-4-phenylpiperazin-1-ium) was chosen asmodel compound. With the aim of improving its potency and establishingnew/additional putative interactions within the α7 binding pocket, twosets of new derivatives were designed and synthesized. The first one wasobtained by introducing different substituents in various positions ofthe phenyl ring and the second by replacing the piperazine ring with thepiperidine nucleus.

The new synthesized derivatives were tested in electrophysiologicalassays on Xenopus oocytes transfected with human α7 cDNA to assess theirfunctional profile at the α7 nAChR subtype and to study their activityas α7 PAMs or silent agonists.

Potential Silent Agonist derivatives: diEPP derivatives synthesized werepartial agonists or inactive at the α7 nAChR subtype. diEPP 17, 19, 20and 21 were the most advantageous silent agonists of the series,producing big responses when co-applied with PNU-120596. Polarinteractions have been hypothesized to be the key to silent agonism.diEPP 17 does not activate α3β4 or α4β2 nAChR, but it is anon-competitive antagonist of these receptor subtypes with IC50 valuesin the range of 10-100 μM. diEPP 19, 20, 21 do not activate α3β4 or α4β2nAChR, but diEPP 19 and 21 are non-competitive antagonists of thesereceptor subtypes.Silent Agonism: Silent agonists are compounds that do not produce asignificant net-charge response of the receptor when applied alone, butdo produce a response when co-applied with a PAM such as PNU-120596.While not wishing to be bound by any one theory, it is possible that thebound state of the receptor in complex with a silent agonist isdesensitized, as revealed by co-application with a PAM. Compared to theagonist bound state, the silent agonist bound state is destabilized withrespect to entry into the conductive O* state (left), and stabilizedwith respect to entry into the PAM sensitive Ds state (right).Compound syntheses: The general approach used for the synthesis of diEPPderivatives 2 is depicted in Scheme 1 and proceeds in two steps. The keystep in the synthesis is a copper-catalyzed Ullmann-type aryl amination(Ma et al., (2003) Org. Lett. 5: 2453-2455) promoted and accelerated bythe α-amino acid L-proline. Even though N-methylglycine was reportedmore effective than L-proline, it was also more reactive toward couplingwith aryl halides, so L-proline was selected as the ligand (Ma et al.,(2003) Org. Lett. 5: 2453-2455). The catalytic coupling allowed avoidingtypical drawbacks such as stoichiometric amounts of copper reagents andthe high costs for palladium catalysts and their phosphine ligands(Zhang et al., (2005) J. Org. Chem. 70: 5164-5173). Both electron-richand electron-deficient aryl halides were successfully coupled with1-ethylpiperazine at 90° C. in DMSO using 10 mol % CuI and 20 mol %L-proline as the catalytic system (Zhang et al., (2005) J. Org. Chem.70: 5164-5173). Generally, the coupling reactions proceeded smoothly,but the range of yields of this key step was quite wide (from 5 to 83%).

The coupling reaction was found to be inhibited by ortho-substituents(compounds 1h and 1j), presumably due to steric interference within theintermediate addition and eliminations in the copper complex; moreover,aryl iodides gave better yields (52-83%) than aryl bromides (20-64%)discounting the ortho-substituted halides. Though aryl bromides wereusually less costly, aryl iodides were therefore first choice wheneverpossible. Once obtained, the N-phenyl-N′-ethyl piperazines 1 wereconverted into the quaternary ammonium salts 2 by reacting them withethyl iodide in tetrahydrofuran and then purified by chromatographyand/or recrystallization to afford the final compounds as purecrystalline products (yields ranged from 15-76%). In a few cases, thecrystallization resulted in formation of co-crystals between the desiredcompound and the crystallization solvent, so that solvent was found tobe present in the final compound even after prolonged evaporation undervacuum. In two cases, we were unable to recrystallize compounds 2n(p-trifluoromethyl) and 2s (meta-fluoro) despite a broad survey ofdifferent solvent systems. The para- and meta-carboxamides 2t and 2u(Table 3) were readily obtained by hydration of the correspondingnitrile diEPP derivatives (2b and 2i) by reacting them with acetaldoximein the presence of tetrakis Pd(0) as catalyst. In this reaction,acetaldoxime acts as an efficient water donor for delivery to thenitrile. That, together with its commercial availability at low price,and the easy separation of product from the side products (acetonitrileand acetamide), made acetaldoxime the most advantageous reagent for thistransformation.

The synthetic routes to diEPP analogues 6 and 8 are shown in FIG. 2.Compound 6, 1,1-diethyl-4-(phenylamino)piperidinium iodide, wassynthesized by N-alkylation of the protected reductive amination productof 1-ethyl-4-piperidone with aniline (FIG. 2). The reductive aminationproceeded in fair yield (%) as a one-pot reaction in dry methanol/AcOHusing sodium cyanoborohydride as reducing agent to afford 3. Completeconsumption of the starting materials was not obtained, even afterhaving heated the reaction to reflux. Attempts to optimize the reactionvia isolation of the imine intermediate, or use of different anhydroussolvents (CH₂Cl₂, toluene, THF), and water-scavenging agents like MgSO₄,with or without acetic acid addition to the reaction mixture, wereunsuccessful.

Compound 3 was then protected at the aromatic amino group by reactionwith trifluoroacetic anhydride in triethylamine in dry dichloromethaneat 0° C. Compound 4 was reacted with iodoethane in THF dry to afford thequaternary ammonium corresponding derivative 5, which was then refluxedin a mixture of methanol-water in the presence of potassium carbonate tocleave the trifluoroacetyl protecting group to produce 6. The4-benzylidine substituted piperidinium salt 8 was obtained after atwo-step synthesis. The key step utilized Wittig-Horner reaction of1-ethyl-4-piperidone and diethyl benzylphosphonate, both commerciallyavailable, in the presence of sodium hydride and [15-crown-5] to providethe alkene intermediate 7 in 45% yield. It was found that use of thecrown ether in the reaction is important to accelerate the reaction;because omission of the crown ether yielded an incomplete reaction after3 days.

After purification, the olefinated product was ethylated to thecorresponding quaternary ammonium salt (8) by reacting it withiodoethane in dry THF, affording, after careful silica chromatographyand recrystallization, the desired final product in 3% yield. This pooryield was attributed to the difficulty in removing an unidentifiedimpurity that, even if present in small amount, co-eluted with thedesired product.

Electrophysiology: The panel of compounds synthesized was assayed usinghuman α7 nAChR expressed in Xenopus oocytes and two-electrode voltageclamping. The profile of compound activity is presented in Table 1. Thepartial agonism activity was evaluated, quantified as net-charge for 30μM applications of compounds, measured relative to the control responseto applications of 60 μM ACh. The induction of PAM-sensitivedesensitization was detected as the net-charge responses when compoundswere co-applied with the PAM PNU-120596 (30 μM compound+10 μMPNU-120596), measured relative to responses to 60 μM ACh applied alone.It is evident that the diEPP series of compounds 2 (structures, shown inTable 3) exhibit an exceptional sensitivity to the nature and positionof the aryl substituent, as evidenced by the broad range of activitiesagainst the α7 nAChR, which included partial agonism, varying degrees ofsilent agonism, and compounds that were effectively inactive, with noagonism and very weak silent agonism.Partial agonists: FIG. 3A presents a summary of compounds, o-chloro(2j),o-methyl(2h), m-hydroxy(2k), naphthalene(2m), p-chloro(2e) andp-cyano(2b), that were classified as partial agonists based on theirability to stimulate a net-charge response that was greater than athreshold value of 1/10th the ACh control, when the compounds wereapplied alone (FIG. 3B). It is a unique property of α7 orthostericagonist-induced currents that they vary systematically in therelationship between peak current and net charge as a function of theeffective concentration applied (Papke & Papke (2002) Br. J. Pharm. 137:49-61; Papke & Thinschmidt (1998) Neurosci. Let. 256: 163-166). This wasdue to the concentration-dependent desensitization of the receptors,which is likely to be associated with high levels of agonist bindingsite occupancy (Williams et al., (2011) J. Gen. Physiol. 137: 369-384)and is observed even when agonists are rapidly applied to small cells(Williams et al., (2012) Mol. Pharmacol. 82: 746-759) or acutelydissociated neurons (Uteshev et al., (2002) Brain Res. 948: 33-46). Thecontrol ACh concentration used for these experiments is roughly the EC80for the net-charge responses.

By normalizing both the peak currents and net-charge measurements of thedrug responses at the probe concentration of 30 μM (FIG. 3B) to those ofthe ACh control, the relationship between the probe concentration andthe EC for each of the drugs was estimated (Papke, R. L. (2005) LifeSci.). At a concentration where the ratio of the normalized measuresequaled one, the drug would be at the same effective concentration as 60μM ACh.

As shown in Table 2, the peak-current-to-net-charge ratio for each ofthe experimental drugs at 30 μM was less than one. Responses to 2m and2e had the highest peak currents to net charge ratios, indicating thatthey are the most potent of the drugs tested. The estimated rank potencyfor the other compounds is given in Table 2. Potency and efficacy can,of course, vary independently. Compounds that that are likely to berelatively potent (based on peak-current-to-net-charge ratios) butproduced relatively small net-charge responses at the test concentrationare likely to be less efficacious than less potent compounds such as 2hand 2j, which produced larger responses at the test concentration. Basedon these considerations, estimated rank efficacies are also provided inTable 2.

Of the partial agonists, the ortho-substituted ones showed the highestresponses, whether non-polar (Me) or polar (Cl). It is evident that,while these compounds were weak as agonists, their response toapplication with PNU-120596 was quite strong (FIG. 3C).

Silent agonists: Several substitutions yielded compounds that werecandidate silent agonists with little or no partial agonism (i.e. aboveour detection threshold, but too small for accurate characterization),comparable to, or perhaps slightly greater than the unsubstituted parentdiEPP compound (Table 1, and FIGS. 4A-4B 5): m-trifluoromethyl (2o),p-methyl(2a), m-chloro(2f), m-bromo(2p), p-methoxy(2c), p-fluoro(2r), p-and m-carboxamide (2t, 2u), and p-trifluoromethyl(2n). However, fourcompounds showed no significant activity as either partial agonists oras silent agonists at the concentration tested: m-methoxy (2g), m-methyl(2d), m-fluoro (2s) and m-cyano (2i) derivatives.

Two analogs of diEPP were also prepared (FIG. 2) in which the N-aryllinkage was modified to test the importance of this position. One,compound 6, replaced the piperazine ring with a piperidine ring linkedto an aniline group; the other completely eliminated the anilinenitrogen, replacing the aniline group with a benzylidine residue,compound 8 (FIG. 2). Compounds 6 and 8 both showed enhancedPAM-dependent responses relative to the parent diEPP compound (FIG. 5),suggesting that the nitrogen atom of the N-aryl bond is not essentialfor silent agonist activity within the diEPP framework.

For some quaternary ammonium diEPP derivatives shown to be silentagonists, the activity of the corresponding tertiary amine was alsoinvestigated, i.e. select ethyl phenyl piperazine derivatives, compound1, Table 3. Specifically, the meta-bromo, meta-chloro,para-trifluoromethyl and para-fluoro compounds (if, 1p, in, 1r) wereexamined. Interestingly, the tertiary amine 1p (meta-bromo) showed anapproximately 5-fold enhanced response relative to diEPP and was onlyreduced by 0.69-fold relative to its corresponding quaternary salt 2p.This result indicated the hard positive charge is not an absoluterequirement for silent agonist activity. With regard to receptor subtypespecificity, none of the compounds tested were partial agonists forα3β4, with only one (2g) showing significant antagonism at 100 μM.Select compounds (2p, 2n) were also screened against α4β2 and foundneither to be partial agonists nor to have significant antagonistactivity at 100 μM.

Other advantageous embodiments of the compositions of the disclosureinclude 5, 6, and 7 cycloalkyl-piperidines, an example of which, and itssynthesis are given in Example 56.

Aryl substitution patterns control state selectivity: Hypothetically, anoptimized silent agonist, by definition, would be a compound thatproduced no detectable channel activation, and would exclusivelystabilize the receptor into a desensitized state or states that maymediate important forms of signal transduction (Papke et al., (2015)NeuroPharm. 91: 34-42; van Maanen et al., (2015) PLOS ONE 10: 1-20). Itwas possible to detect desensitized states (Ds) that are sensitive to,and become conductive when the receptor is treated with a PAM. With thisin mind, a compound being a good silent agonist implies that it producesa robust response when co-applied with PNU-120596, and shows little (ifany) ability to activate the receptor on its own.

With regard to discerning a pharmacophore for silent agonists, it mustbe noted that some substituents could have a greater effect on thediminution of agonism, or others might have a greater effect oninduction and stabilization of desensitization. In some cases these twoeffects may work together as is evident by the data shown in FIGS.3A-3C, where, relative to the parent compound diEPP, some substituentswere able to facilitate both a conductive state and a non-conductive Dsstate.

Starting from diEPP as the parent compound, through differentmodifications of the aryl ring, it was possible to obtain severalderivatives with enhanced silent agonism profiles (defined as largePAM-dependent currents without increased orthosteric agonism). Among theset of diEPP derivatives generated, the best compounds were meta- orpara-substituted diEPP, with small-to-medium size substituent groups, inparticular the para-trifluoromethyl, para-fluoro, para- andmeta-carboxamide derivatives.

One question was whether a single common property of these functionalgroups might serve to explain their ability to enhance silent agonism.Fluorine atoms, the trifluoromethyl group, and carboxamides can be quitedifferent in the nature of their interactions with protein bindingsites, but in our case they all had enhanced silent agonism behaviorcompared to the parent compound diEPP, more so than other substituents.To interpret this observation, different atomic properties and atomicinteractions were considered. Taking into account the polarity of thegroup as a primary feature, it is ascribable to fluorine atoms in thefirst two cases and to the oxygen/amino groups in the latter two.However, not all compounds containing polar groups showed great silentagonism; for example the methoxy and hydroxy derivatives (Table 1), sothat polarity on its own is insufficient to explain in a simple way theactivity of the best silent agonists. In some cases, fluorine and thetrifluoromethyl groups have been considered to enhance lipophilicity(Bohm et al., (2004) Chembiochem. 5: 637-643), and if that is the casein our study, introduction of lipophilic substituents such as the methylgroup might be expected to improve silent agonism compared to the parentcompound diEPP. However, para-methyl and meta-methyl derivatives bothfailed to effectively induce the Ds state, which supports the idea thatpolar interactions are operative.

Although hydrogen bonds are well-known to be involved in a myriad ofprotein-ligand interactions, fluorine rarely participates as ahydrogen-bond acceptor and it would be a weak interaction (Zhou et al.,(2009) J. Chem. Inf. Model 49: 2344-2355); however, carboxamides areknown to be good H-bond acceptors. So if hydrogen bonding was the keyinteraction behind the silent agonism improvement of those selectivecompounds compared to diEPP, carboxamide derivatives would show a muchgreater improvement than the CF₃- and F-derivatives, but this was notobserved. Thus, the basis for enhanced silent agonism of fluoro,trifluoromethyl, and carboxamide residues may be multifactorial butshare in common their ability to stabilize an overall conformationalstate of the receptor that is desensitized yet sensitive to PNU-120596through a variety of point-to-point interactions between the variouscompounds and elements in the silent agonist binding site, considered tobe an extension of the site where typical orthosteric agonists such asACh bind. Intriguing results were observed for the meta-substitutedhalogen-containing diEPP derivatives, which for the fluoro, chloro, andbromo derivatives showed increasing potentiated responses (Table 1).

To describe these results, halogen bonding interactions were considered.The strength of the interaction increases in the orderfluorine<chlorine<bromine<iodine. In our meta-substituted derivatives,an increase in the PAM-dependent currents was observed (FIG. 4A), movingfrom fluorine (2s; 0.2-fold relative to ACh) hydrogen (diEPP; 1.33-fold)to chlorine (2f; 4.23-fold) to bromine (2p; 9.98-fold), consistent withhalogen-bonding interaction with a suitable electron-donor partner inthe binding site of the receptor. Indeed, we would predict thatelectron-rich or electrondonating meta substituents might perform poorlyas silent agonists, and this was the case. The meta-methoxy andmeta-cyano diEPP (2g, i) derivatives, respectively showed 50% andequivalent PAM-dependent responses compared to the parent unsubstituteddiEPP compound. Interestingly, the meta-hydroxy derivative, capable ofacting as a hydrogen bond donor, was found to be a partial agonist,suggesting that a unique hydrogen bond at the phenolic OH-group inducesa conductive state of the receptor. Chlorine or bromine substitutions inthe para or ortho positions do not yield silent agonists, since, infact, para-chloro, para-bromo and ortho-chloro derivatives showedpartial agonist activities with the α7 nAChR subtype (Table 1, FIGS.3A-3C).

However, in contrast with the trend highlighted among the para-halogendiEPPs, para-fluoro and para-trifluoromethyl diEPP are two of the mostactive compounds as silent agonists in the diEPP series. The explanationfor the divergence in the activity of these para substituents mustreside in the unique behavior of fluorine substituents, but this is acomplex interplay of numerous effects (Zhou et al., (2009) J. Chem. Inf.Model 49: 2344-2355), and in lieu of a high-resolution structure,becomes a speculative endeavor to determine its origin.

Both ortho-chloro and ortho-methyl derivatives are partial agonists ofthe α7 receptor, and these two substituents have similarConnolly-excluded molecular volumes (14.3 Å versus 16.9 Å), suggestingthat a steric directing effect of an ortho-substituent may facilitateentry of the receptor into a conductive state. Yet, the o,p-dimethoxyanalogue 21 is not a partial agonist, suggesting that thepara-substituent may supersede the putative agonism-promoting effect ofan ortho substituent.

The naphthalene derivative (2m) was intended to investigate thetolerance of the binding pocket of the α7 receptor towards more bulkygroups, and it became a partial agonist compared to the parent diEPP.These data, compared with the activity of ortho substituted compounds,suggest that the extended point-to-point interactions between the largernaphthalene ligand and receptor are yet another way to induce conductivestates of the receptor in addition to the internal conformationalbiasing or ortho substituents. The relationship between the steric bulkaround the core ammonium group as a way to convert partial agonists intosilent agonists has been discussed (Papke et al., (2014) J. Pharmacol.Experimental Therap. 350: 665-680). Here, substitutions of the aromaticring are remote from the core ammonium group and do not appear to followa simple correlation of substituent bulk with silent agonism. Thus,aromatic substituents on diEPP compounds appear to be modulating silentagonism in a different way than simple ammonium compounds do.

Modifications at the core piperazine nitrogen atoms: As part of the workto dissect structural features within the diEPP silent agonistpharmacophore, the importance of the nitrogen in the piperazine ringthat was attached to the phenyl group was tested and if smallmodifications at this point in the molecule might enhance silentagonism. Both compounds 6 and 8 were enhanced in terms of theirresponses when co-applied with PNU-120596, though compound 6 wassuperior to compound 8 from the point of view of the ratio of the amountof desensitization to residual partial agonism. While we were able tomeasure currents on application of 8 to α7, application of 6 to α7resulted in no channel activation within experimental error. Compound 6thus may serve as a suitable framework for development of cleaner, morestate-selective silent agonists.

The phenyl ethyl piperazine (PEP) compounds if, in, 1p, 1r were selectedfor testing because they correspond to diethylammonium compounds 2 thathad significant ability to enter D_(s) as evidenced by strong PAMco-application responses. It was asked if the hard positive chargepresent in active compounds 2 was required for silent agonism (FIG. 4C).It was found that, for the most part, these compounds were inactive,with no partial agonism and weak responses to PAM co-application. Theexception was meta-bromo PEP 1p, which had a response nearlyindistinguishable from the diethyl version 2p (6.9±2.4 vs 10.0±1.7,Table 1). This result provides another indication that the core minimalammonium pharmacophore observed for simple ammonium compounds may not berequired if suitable structural features remote from the core chargednitrogen are present.

This observation is of particular importance for the potentialtherapeutic development of silent agonists that will be more likely tocross the blood-brain barrier. However, the generally greater activityof the quaternary amines can be exploited for specific indications thatwould not require brain penetration, such as the targeting of peripheralimmune cells for anti-inflammatory activity.

A critical question speaks to the mechanism by which a bound ligand isable to facilitate entry into a desensitized state or states. Availabledata indicate that silent agonists do not precisely place the receptorinto a single desensitized state (Williams et al., (2011) Mol.Pharmacol. 80: 1013-1032). Rather, the receptor silent agonist complexis in a highly dynamic series of states; depending on occupancy levelsand application time, the complex can enter PAM-insensitive desensitizedstates (D_(i)), which may evolve over time into PAM-sensitivedesensitized states (D_(s)) and or eventually dissociate from thereceptor (Papke et al., (2015) NeuroPharm. 91: 34-42). Based on theresults described here, it is entirely reasonable that a number ofdifferent amino acid side chains in the binding site may be utilized asinteracting partners for selective entry into a D_(s) state. Moleculardocking studies suggested that multiple binding poses in the orthostericagonist binding site of the receptor are possible, which leads to anumber of hypotheses about specific ligand receptor interactions ofinterest.

The diEPP molecular framework has been an excellent platform to identifysome enhanced silent agonists as well as to identify new molecularfeatures that regulate different features associated with silentagonism. It has been found the p-fluoro and p-trifluoromethyl, and p-and m-carboxamide groups to provide strongly enhanced entry of α7 intothe D_(s) state relative to the parent unsubstituted compound diEPP, anddo so with little activity as partial agonists. The limited number ofortho substitutions tested yielded partial agonists. When the paraposition was substituted with Cl, Br, or cyano, the compounds werepartial agonists with strong responses to PAM co-application; however,methoxy, fluoro, carboxamide, and trifluoromethyl substituents haddiminished partial agonism and strong PAM co-application responses. Themeta position is distinguished from para by the fact that fewersubstitutions lead to partial agonism (meta-hydroxyl compound 2k being anoteworthy exception).

The PAM co-application responses were low for most meta-group compounds,with the bromo and carboxamide being standouts for their strong D_(s)response to PAM application. Interestingly, the nitrogen of the N-aryllinkage is not required for activity, as exemplified by compound 8,indicating the N-aryl functionality is not a critical determinant forstabilizing desensitization. Finally, while three of the fournon-quaternary compounds 1 were inactive as silent agonists, themeta-bromo compound 1p showed modest silent agonism with littlediminution relative to its ammonium analog 2p, demonstrating that a hardpositive charge is not required for activity. These compounds couldprove interesting for applications requiring blood brain barrierpermeability.

Thus, the Ullman-type couplings used to synthesize the diEPP compoundsproved to be versatile for a variety of substituted phenyl halides.Unsurprisingly, couplings of ortho-substituted aryl halides to ethylpiperazine often produced significantly lowered yields. However, becausewe found that the most effective silent agonists were notortho-substituted this synthetic limitation had no impact on this study.In the meta series, the most effective silent agonist was thecarboxamide-substituted diEPP, with a sharp demarcation from othercompounds. In the para series, this demarcation was less sharp, withp-CF3 being most active, closely followed by the p-carboxamide. The paraposition appears more tolerant of the nature of the substituent. Aquaternary center is not absolutely required for silent agonism, asexemplified by compound 1p, bearing a single ethyl group. Compounds 6and 8 which are diEPP analogs that lack the piperazine ring are alsomoderately active, providing a demonstration that the diEPP platform mayprovide an excellent opportunity to flexibly explore chemical space.

Accordingly, one aspect of the disclosure encompasses embodiments of acompound having the formula I, II, III, or IV:

or a pharmaceutically acceptable salt thereof, wherein:

Y can be:

Z can be

n can be 1, 2, 3, or 4; R₁, R₂, and R₃ can each be independently ahydrogen, an alkyl group, cyano, an alkoxy group, a halogen, atrihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl, andwherein the halogen is fluorine, chlorine or bromine; R₄ can be ahydrogen or an ethyl group; and R_(s) can be a hydrogen or acarboxytrifluoromethyl.

In some embodiments of this aspect of the disclosure, R₁, R₂, and R₃ caneach be independently a hydrogen, a methyl, cyano, methoxy, a halogen,trihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl.

In some embodiments of this aspect of the disclosure, R₁, R₂, and R₃ caneach be independently a hydrogen, a methyl, cyano, methoxy, a halogen, atrihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl, and R_(s)is hydrogen.

In some embodiments of this aspect of the disclosure, when R₁ is amethyl or a CN, R₂ and R₃ are each hydrogen; when R₁ is a methoxy, R₂ ishydrogen R₃ is hydrogen or methoxy; when R₁ is a halogen, R₂ and R₃ areeach hydrogen; when R₁ is a trifluoromethyl, R₂ and R₃ are eachhydrogen; when R₁ is a carboxamide, pentafluorosulfanyl, R₂ and R₃ areeach hydrogen; when R₁ and R₃ are each hydrogen, R₂ is a methyl, a CN, amethoxy, a halogen, a trifluoromethyl, or a carboxamide, or OH; when R₁and R₂ are each hydrogen, R₃ is a methyl or Cl; and R₄ is an ethylgroup.

Another aspect of the disclosure encompasses embodiments of apharmaceutical composition comprising a compound having the formula I,II, III, or IV:

or a pharmaceutically acceptable salt thereof, wherein:

Y can be:

Z can be

n can be 1, 2, 3, or 4; R₁, R₂, and R₃ can each be independently ahydrogen, an alkyl group, cyano, an alkoxy group, a halogen, atrihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl, andwherein the halogen is fluorine, chlorine or bromine; R₄ can be ahydrogen or an ethyl group; and R₅ can be a hydrogen or acarboxytrifluoromethyl; and a pharmaceutically acceptable carrier.

In some embodiments of this aspect of the disclosure, R₁, R₂, and R₃ caneach be independently a hydrogen, a methyl, cyano, methoxy, a halogen,trihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl.

In some embodiments of this aspect of the disclosure, R₁, R₂, and R₃ caneach be independently a hydrogen, a methyl, cyano, methoxy, a halogen, atrihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl, and R₅ ishydrogen.

In some embodiments of this aspect of the disclosure, when R₁ is amethyl or a CN, R₂ and R₃ are each hydrogen; when R₁ is a methoxy, R₂ ishydrogen R₃ is hydrogen or methoxy; when R₁ is a halogen, R₂ and R₃ areeach hydrogen; when R₁ is a trifluoromethyl, R₂ and R₃ are eachhydrogen; when R₁ is a carboxamide, pentafluorosulfanyl, R₂ and R₃ areeach hydrogen; when R₁ and R₃ are each hydrogen, R₂ is a methyl, a CN, amethoxy, a halogen, a trifluoromethyl, or a carboxamide, or OH; when R₁and R₂ are each hydrogen, R₃ is a methyl or Cl; and R₄ is an ethylgroup.

In some embodiments of this aspect of the disclosure, the compositioncan be formulated to deliver to a human or animal subject in needthereof, an amount of the compound therapeutically effective inmodulating the activity of a nicotinic acetylcholine receptor in therecipient patient, and wherein the therapeutically effective amount candelivered as a single dose or as a series of doses.

In some embodiments of this aspect of the disclosure, the nicotinicacetylcholine receptor positive allosteric modulator (PAM) can be a typeII PAM.

In some embodiments of this aspect of the disclosure, the nicotinicacetylcholine receptor positive allosteric modulator (PAM) can be thetype II PAM1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea(PNU-120596).

Yet another aspect of the disclosure encompasses a method of modulatingthe activity of a nicotinic acetylcholine receptor in an animal or humansubject by administering to said subject therapeutically effective dosesof a silent agonist of the nicotinic acetylcholine receptor and anicotinic acetylcholine receptor positive allosteric modulator (PAM).

In some embodiments of this aspect of the disclosure, the silent agonistof the nicotinic acetylcholine receptor and the nicotinic acetylcholinereceptor positive allosteric modulator (PAM) can be administered to thesubject simultaneously or as consecutive doses.

In some embodiments of this aspect of the disclosure, the silent agonistof the nicotinic acetylcholine receptor is a compound having the formulaI, II, III, or IV:

or a pharmaceutically acceptable salt thereof, wherein:

Y can be:

Z can be

n can be 1, 2, 3, or 4; R₁, R₂, and R₃ can each be independently ahydrogen, an alkyl group, cyano, an alkoxy group, a halogen, atrihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl, andwherein the halogen is fluorine, chlorine or bromine; R₄ is a hydrogenor an ethyl group; and R₅ is a hydrogen or a carboxytrifluoromethyl.

In some embodiments of this aspect of the disclosure, the nicotinicacetylcholine receptor positive allosteric modulator (PAM) can be a typeII PAM.

In some embodiments of this aspect of the disclosure, the nicotinicacetylcholine receptor positive allosteric modulator (PAM) can be thetype II PAM1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea(PNU-120596).

In some embodiments of this aspect of the disclosure, the compositioncan be formulated to deliver to a human or animal subject in needthereof, an amount of the compound therapeutically effective inmodulating the activity of a nicotinic acetylcholine receptor in therecipient patient, and wherein the therapeutically effective amount isdelivered as a single dose or as a series of doses.

It should be emphasized that the embodiments of the present disclosure,particularly any “preferred” embodiments, are merely possible examplesof the implementations, merely set forth for a clear understanding ofthe principles of the disclosure. Many variations and modifications maybe made to the above-described embodiment(s) of the disclosure withoutdeparting substantially from the spirit and principles of thedisclosure. All such modifications and variations are intended to beincluded herein within the scope of this disclosure, and protected bythe following claims.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentdisclosure to its fullest extent. All publications recited herein arehereby incorporated by reference in their entirety.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range. The term “about” can include 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, or +10%, or more of the numerical value(s) being modified.

EXAMPLES Example 1

Reagents for chemical synthesis were purchased from Fisher Scientific(Pittsburgh, Pa.), Sigma-Aldrich (St. Louis, Mo.), or Tokyo ChemicalIndustry (TCI America, Portland, Oreg.). Melting points (uncorrected)were obtained on an MFB-595010M Gallenkamp apparatus equipped with adigital thermometer.

NMR spectra (¹H and ¹³C) were recorded on a Varian Mercury-300 (300 and75.0 MHz, respectively) or Varian Inova-500 (5 and 126.0 MHz)instruments using CDCl₃, CD₃OD, (CD₃)₂CO, or (CD₃)₂SO as solvent.Chemical shifts (δ scale) are reported in parts per million (ppm)relative to the peak of the internal standard TMS (δ=0.00 ppm) forCDCl₃, CD₃OD, (CD₃)₂CO or relative to the central peak of the solvent(δ=2.20 ppm for (CD₃)₂SO) in ¹H NMR and relative to the central peak ofthe solvent (δ=77.16 ppm for CDCl₃, 49.00 for CD₃OD, 39.52 for (CD₃)₂SO(DMSO-d6), and 29.84 for (CD₃)₂CO in ¹³C NMR. Processing of the spectrawas performed with MestReNova 8.1.1.

Mass spectra were obtained on a Hewlett-Packard 5988A spectrometer or onan Agilent 6220 ESI TOF (Santa Clara, Calif.) mass spectrometer equippedwith electrospray and DART sources operated in positive ion mode.

Column chromatography was performed with silica gel (Sigma-Aldrich,230-4mesh) or neutral alumina. Reactions were monitored by TLC using0.25 mm silica gel F-254 glass plates (EMD Millipore or neutral aluminaTLC plates).

All reagents were of reagent quality or were purified before use.Organic solvents were of analytical grade or were purified by standardprocedures. Reactions were carried out in flame-dried glassware andunder argon atmosphere when required. In those cases, anhydrous solventswere used in the reactions. Compound purity was 95% as determined by ¹HNMR analyses.

Example 2

General procedure A for the coupling reaction of aryl halides with1-ethylpiperazine to produce N-aryl piperazines 1a-1s: In a flame-driedand argon-flushed round-bottom flask, a mixture of aryl halide (1equiv), N-ethylpiperazine (1 equiv), K₂CO₃ (2 equiv, for aryl iodide) orK₃PO₄ (2 equiv, for aryl bromide), CuI (0 equiv), and L-proline (0.2equiv) in dry DMSO (1.6 mL/mmol 1-ethylpiperazine) was heated at 90-100°C. until completion (TLC in hexanes/ethyl acetate 9/1).

To the cooled mixture was then added deionized water, the organic layerwas separated, and the aqueous layer was extracted three times withethyl acetate. The combined organic layers were washed with saturatedbrine, dried over MgSO₄, and concentrated in vacuo. The residual oil waspurified on a silica gel column eluted with a mixture of ethylacetate/methanol to afford the corresponding aniline. When coupling arylbromides, 1-ethylpiperazine and the aryl bromide were used in a 1:1stoichiometric ratio.

Example 3

1-ethyl-4-p-tolylpiperazine (1a) was obtained by reaction of4-iodotoluene (114 mg, 0.53 mmol, 1 equiv) and 1-ethylpiperazine (100μL, 0.79 mmol, 1.5 equiv) (orange oil; 70 mg, 65% yield); R_(f)=0.51 inCH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz): δ 6.99 (d, J=8.2 Hz, 2H), 6.77(d, J=8.3 Hz, 2H), 3.14-3.06 (m, 4H), 2.59-2.49 (m, 4H), 2.40 (q, J=7.2Hz, 2H), 2.18 (s, 3H), 1.05 (t, J=7.2 Hz, 3H). ¹³C NMR (CDCl₃, 75 MHz) δ149.3, 129.7, 129.2, 116.5, 53.0, 52.4, 49.7, 20.5, 12.0.

Example 4

4-(4-ethylpiperazin-1-yl)benzonitrile (1b) was obtained by reaction of4-bromobenzonitrile (191 mg, 1.05 mmol, 1 equiv) and 1-ethylpiperazine(200 μL, 1 .mmol, 1.5 equiv) (orange oil; 142 mg, 63% yield); R_(f)=0.48in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz): δ 7.41 (d, J=8.8 Hz, 1H),6.79 (d, J=9.3 Hz, 1H), 3.31-3.25 (m, 4H), 2.55-2.49 (m, 4H), 2.41 (q,J=7.2 Hz, 2H), 1.06 (t, J=7.2 Hz, 3H). ¹³C NMR (CDCl₃, 75 MHz): δ 153.4,133.5, 120.2, 114.2, 100.2, 52.4, 52.4, 47.2, 12.0. HMRS [M+H]⁺(C₁₃H₁₈N₃) Calcd 216.1495, Found 216.1499.

Example 5

1-ethyl-4-(4-methoxyphenyl)piperazine (1c) was obtained by reaction of4-iodoanisole (616 mg, 2.63 mmol, 1 equiv) and 1-ethylpiperazine (500μL, 3.94 mmol, 1.5 equiv) (pale yellow solid; 302 mg, 52% yield);m.p.=56.6-57.8° C.; R_(f)=0.44 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300MHz): δ 6.84 (d, J=9.1 Hz, 2H), 6.77 (d, J=9.2 Hz, 2H), 3.70 (s, 3H),3.09-3.02 (m, 4H), 2.60-2.52 (m, 4H), 2.41 (q, J=7.3 Hz, 2H), 1.06 (t,J=7.2 Hz, 3H). ¹³C NMR (CDCl₃, 75 MHz): δ 153.7, 145.7, 118.1, 114.4,55.5, 52.9, 52.3, 50.6, 12.0.

Example 6

1-ethyl-4-m-tolylpiperazine (1d) was obtained by reaction of3-bromotoluene (320 μL, 450 mg, 2.63 mmol, 1 equiv) and1-ethylpiperazine (500 μL, 3.94 mmol, 1.5 equiv) (yellow oil; 106 mg,20% yield); R_(f)=0.51 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz): δ7.15 (tt, J=8.1, 1.1 Hz, 1H), 6.78-6.74 (m, 2H), 6.74-6.66 (m, 2H),3.26-3.18 (m, 4H), 2.66-2.59 (m, 4H), 2.49 (q, J=7.2 Hz, 2H), 2.31 (s,3H), 1.13 (t, J=7 .Hz, 3H). ¹³C NMR (CDCl₃, 75 MHz): δ 151.5, 138.9,129.0, 120.7, 117.0, 113.3, 52.9, 52.4, 49.2, 21.9, 12.0.

Example 7

1-(4-chlorophenyl)-4-ethylpiperazine (1e) was obtained by reaction of4-chlorobromobenzene (504 mg, 2.63 mmol, 1 equiv) and 1-ethylpiperazine(500 μL, 3.94 mmol, 1.5 equiv) (pale orange oil; 146 mg, 25% yield);R_(f)=0.55 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz): δ 7.13 (d, J=9.1Hz, 1H), 6.77 (d, J=9.0 Hz, 2H), 3.15-3.08 (m, 4H), 2.57-2.51 (m, 4H),2.41 (q, J=7.2 Hz, 3H), 1.06 (t, J=7.2 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃):δ 150.1, 129.1, 124.6, 117.3, 52.8, 52.5, 49.3, 12.1. HMRS [M+H]⁺(C₁₂H₁₈ClN₂) Calcd 225.1153, Found 225.1155.

Example 8

1-(3-chlorophenyl)-4-ethylpiperazine (1f) was obtained by reaction of1-chloro-3-iodobenzene (1.3 mL, 2.50 g, 10.50 mmol, 1 equiv) and1-ethylpiperazine (2.0 mL, 15.75 mmol, 1.5 equiv) (orange oil; 1.96 g,83% yield); R_(f)=0.53 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz): δ7.15 (t, J=8.1 Hz, 1H), 6.88 (t, J=2.2 Hz, 1H), 6.82-6.75 (m, 2H),3.25-3.19 (m, 4H), 2.62-2.56 (m, 4H), 2.47 (q, J=7.2 Hz, 2H), 1.13 (t,J=7.2 Hz, 3H). ¹³C NMR (CDCl₃, 75 MHz): δ 152.4, 135.0, 130.0, 119.2,115.7, 113.8, 52.7, 52.4, 48.7, 12.1. HMRS [M+H]⁺ (C₁₂H₁₈ClN₂) Calcd225.1153, Found 225.1149.

Example 9

1-ethyl-4-(3-methoxyphenyl)piperazine (1g) was obtained by reaction of3-iodoanisole (1.25 mL, 2.46 g, 10.50 mmol, 1 equiv) and1-ethylpiperazine (2.0 mL, 15.75 mmol, 1.5 equiv) (red oil, 1.62 g, 70%yield); R_(f)=0.50 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz): δ 7.08(t, J=8.2 Hz, 1H), 6.46 (ddd, J=8.3, 2.3, 1.0 Hz, 1H), 6.39 (t, J=2.3Hz, 1H), 6.33 (ddd, J=8.2, 2.3, 0.9 Hz, 1H), 3.70 (s, 3H), 3.17-3.10 (m,4H), 2.55-2.49 (m, 4H), 2.39 (q, J=7.2 Hz, 1H), 1.05 (t, J=7.2 Hz, 3H).¹³C NMR (CDCl₃, 75 MHz): δ 160.7, 152.8, 129.8, 108.9, 104.4, 102.5,55.2, 52.91, 52.4, 49.1, 12.1.

Example 10

1-ethyl-4-o-tolylpiperazine (1h) was obtained by reaction of2-iodotoluene (1.34 mL, 2.29 g, 10.50 mmol, 1 equiv) and1-ethylpiperazine (2.0 mL, 15.75 mmol, 1.5 equiv) (reddish oil; 386 mg,18% yield); R_(f)=0.51 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz): δ7.20-7.12 (m, 2H), 7.06-7.01 (m, 1H), 7.00-6.94 (m, 1H), 3.00-2.94 (m,4H), 2.69-2.57 (m, 4H), 2.51 (q, J=7.3 Hz, 2H), 2.30 (s, 3H), 1.14 (t,J=7.3 Hz, 3H). ¹³C NMR (CDCl₃, 75 MHz): δ 151.6, 132.6, 131.1, 126.6,123.2, 119.1, 53.4, 52.5, 51.7, 18.0, 12.1.

Example 11

3-(4-ethylpiperazin-1-yl)benzonitrile (1i) was obtained by reaction of3-bromobenzonitrile (1.92 g, 10.50 mmol, 1 equiv) and 1-ethylpiperazine(2.0 mL, 15.75 mmol, 1.5 equiv) (pale orange oil; 1.36 g, 60% yield);R_(f)=0.48 in CH₂Cl₂/MeOH 9/1; 1H NMR (CDCl₃, 500 MHz): δ 7.35-7.28 (m,1H), 7.14-7.07 (m, 3H), 3.29-3.22 (m, 4H), 2.65-2.58 (m, 4H), 2.48 (q,J=7.2 Hz, 2H), 1.14 (t, J=7.2 Hz, 3H). ¹³C NMR (CDCl₃, 126 MHz): δ151.5, 130.0, 122.5, 119.9, 119.5, 118.5, 113.2, 52.6, 52.4, 48.5, 12.1.

Example 12

1-(2-chlorophenyl)-4-ethylpiperazine (1j) was obtained by reaction of1-chloro-2-iodobenzene (2.50 g, 10.50 mmol, 1 equiv) and1-ethylpiperazine (2.0 mL, 15.75 mmol, 1.5 equiv) (brown oil; 110 mg, 5%yield); R_(f)=0.53 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz): δ 7.35(dd, J=7.9, 1.5 Hz, 1H), 7.22 (td, J=7.3, 1.5 Hz, 1H), 7.06 (dd, J=8.0,1.6 Hz, 1H), 6.97 (td, J=7.6, 1.5 Hz, 1H), 3.18-3.06 (m, 4H), 2.75-2.62(m, 4H), 2.53 (q, J=7.2 Hz, 2H), 1.14 (t, J=7.2 Hz, 3H). ¹³C NMR (CDCl₃,75 MHz): δ 149.3, 130.7, 128.9, 127.7, 123.8, 120.5, 53.0, 52.4, 51.1,12.0.

Example 13

3-(4-ethylpiperazin-1-yl)phenol (1k) was obtained by reaction of3-iodophenol (2.31 g, 10.50 mmol, 1 equiv) and 1-ethylpiperazine (2.0mL, 15.75 mmol, 1.5 equiv) (brown solid; 703 mg, 32% yield);m.p.=150.3-151.6° C.; R_(f)=0.38 in CH₂Cl₂/MeOH 85/15; ¹H NMR (CD₃OD,300 MHz): δ 7.03 (t, J=8.1 Hz, 1H), 6.45 (ddd, J=8.2, 2.4, 0.9 Hz, 1H),6.40 (t, J=2.3 Hz, 1H), 6.30 (ddd, J=8.0, 2.3, 0.9 Hz, 1H), 3.20-3.11(m, 4H), 2.68-2.57 (m, 4H), 2.49 (q, J=7.2 Hz, 2H), 1.14 (t, J=7.2 Hz,3H). ¹³C NMR (CD₃OD, 75 MHz): δ 159.2, 154.0, 130.8, 109.0, 108.3,104.5, 53.7, 53.3, 50.0, 11.8. HMRS [M+H]⁺ (C₁₂H₁₉N₂O) Calcd 207.1492,Found 207.1496.

Example 14

1-(2,4-dimethoxyphenyl)-4-ethylpiperazine (11) was obtained by reactionof 1-bromo-2,4-dimethoxybenzene (2.28 g, 1.51 mL, 10.5 mmol, 1 equiv)and 1-ethylpiperazine (2.0 mL, 15.75 mmol, 1.5 equiv) (brown oil; 839mg, 32% yield); R_(f)=0.36 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz):δ 6.88 (d, J=8.5 Hz, 1H), 6.48 (d, J=2.6 Hz, 1H), 6.43 (dd, J=8.6, 2.7Hz, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 3.14-2.97 (m, 4H), 2.75-2.57 (m,4H), 2.49 (q, J=7.2 Hz, 2H), 1.13 (t, J=7.2 Hz, 3H). ¹³C NMR (CDCl₃, 75MHz): δ 156.2, 153.5, 135.4, 118.6, 103.4, 100.0, 55.6, 55.5, 53.3,52.5, 51.3, 12.1.

Example 15

1-ethyl-4-(naphthalen-2-yl)piperazine (1m) was obtained by reaction of2-bromonaphthalene (2.17 g, 10.5 mmol, 1 equiv) and 1-ethylpiperazine(2.0 mL, 15.75 mmol, 1.5 equiv) (brownish solid; 1.02 g, 40% yield);m.p.=69.1-70.0° C.; R_(f)=0.38 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300MHz): δ 7.76-7.65 (m, 3H), 7.39 (ddd, J=8.1, 6.8, 1.4 Hz, 1H), 7.33-7.24(m, 2H), 7.12 (d, J=2.5 Hz, 1H), 3.39-3.27 (m, 4H), 2.74-2.62 (m, 4H),2.60 (s, DMSO), 2.51 (q, J=7.2 Hz, 2H), 1.15 (t, J=7.2 Hz, 3H). ¹³C NMR(CDCl₃, 75 MHz): δ 149.3, 134.7, 128.8, 128.6, 127.5, 126.8, 126.3,123.4, 119.4, 110.3, 53.0, 52.5, 49.6, 41.1 (DMSO), 12.2.

Example 16

1-ethyl-4-(4-(trifluoromethyl)phenyl)piperazine (In) was obtained byreaction of 1-iodo-4-(trifluoromethyl)benzene (2.86 g, 1.54 mL, 10.50mmol, 1 equiv) and 1-ethylpiperazine (2.0 mL, 15.75 mmol, 1.5 equiv)(white solid; 2.20 g, 81% yield); m.p.=56.3-56.5° C.; R_(f)=0.51 inCH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz): δ 7.48 (d, J=8.6 Hz, 2H), 6.92(d, J=8.6 Hz, 2H), 3.36-3.26 (m, 4H), 2.64-2.56 (m, 4H), 2.48 (q, J=7.2Hz, 2H), 1.14 (t, J=7.2 Hz, 3H). ¹³C NMR (CDCl₃, 126 MHz): δ 153.45,126.44, 125.0 (q, J=276 Hz) 120.4 (q, J=37 Hz), 114.5, 52.7, 52.5, 48.1,12.1. HMRS [M+H]⁺ (C₁₃H₁₈F₃N₂) Calcd 259.1417, Found 259.1426.

Example 17

1-ethyl-4-(3-(trifluoromethyl)phenyl)piperazine (10) was obtained byreaction of 3-bromobenzotrifluoride (2.36 g, 1.47 mL, 10.50 mmol, 1equiv) and 1-ethylpiperazine (2.0 mL, 15.75 mmol, 1.5 equiv) (yellowoil; 1.73 g, 64% yield); R_(f)=0.54 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃,300 MHz): δ 7.39-7.29 (m, 1H), 7.14-7.10 (m, 1H), 7.09-7.03 (m, 2H),3.31-3.21 (m, 4H), 2.66-2.57 (m, 4H), 2.48 (q, J=7.2 Hz, 2H), 1.13 (t,J=7.2 Hz, 3H). ¹³C NMR (CDCl₃, 126 MHz): δ 151.5, 131.5 (q, J=31 Hz)129.6, 124.4, (q, J=271 Hz) 118.6, 115.7, 112.1, 52.7, 52.4, 48.7, 12.1.HMRS [M+H]⁺ (C₁₃H₁₈F₃N₂) Calcd 259.1417, Found 259.1423.

Example 18

1-(3-bromophenyl)-4-ethylpiperazine (1p) was obtained by reaction of1,3-dibromobenzene (3.72 g, 1.90 mL, 15.75 mmol, 1 equiv) and1-ethylpiperazine (2.0 mL, 15.75 mmol, 1.5 equiv) (reddish oil, 1.60 g,38% yield); R_(f)=0.49 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz): δ7.09 (t, J=8.1 Hz, 1H), 7.05-7.01 (m, 1H), 6.97-6.91 (m, 1H), 6.86-6.79(m, 1H), 3.26-3.16 (m, 4H), 2.63-2.53 (m, 4H), 2.46 (q, J=7.2 Hz, 2H),1.12 (t, J=7.2 Hz, 3H). ¹³C NMR (CDCl₃, 75 MHz): δ 152.5, 130.3, 123.3,122.1, 118.6, 114.3, 52.7, 52.4, 48.7, 12.1. HMRS [M+H]⁺ (C₁₂H₁₈BrN₂)Calcd 269.0648, Found 269.0641.

Example 19

1-(4-bromophenyl)-4-ethylpiperazine (1q) was obtained by reaction of1,4-dibromobenzene (2.48 g, 10.50 mmol, 1 equiv) and 1-ethylpiperazine(1.33 mL, 10.50 mmol, 1.5 equiv) (off-white solid; 913 mg, 32% yield);m.p.=85.0-85.5° C.; R_(f)=0.49 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300MHz): δ 7.33 (d, J=9.0 Hz, 2H), 6.79 (d, J=9.0 Hz, 2H), 3.24-3.13 (m,4H), 2.65-2.55 (m, 4H), 2.47 (q, J=7.2 Hz, 2H), 1.13 (t, J=7.2 Hz, 3H).¹³C NMR (CDCl₃, 75 MHz): δ 150.4, 131.9, 117.6, 111.8, 52.8, 52.4, 49.1,12.1.

Example 20

1-ethyl-4-(4-fluorophenyl)piperazine (1r) was obtained by reaction of4-fluoroiodobenzene (2.33 g, 1.21 mL, 10.50 mmol, 1 equiv) and1-ethylpiperazine (2.0 mL, 15.75 mmol, 1.5 equiv) (yellowish solid; 1.77g, 81% yield); m.p.=30.0-30.5° C.; R_(f)=0.46 in CH₂Cl₂/MeOH 9/1; ¹H NMR(CDCl₃, 300 MHz): δ 7.02-6.92 (m, 2H), 6.92-6.84 (m, 2H), 3.20-3.10 (m,4H), 2.66-2.57 (m, 4H), 2.48 (q, J=7.3 Hz, 2H), 1.13 (t, J=7.2 Hz, 3H).¹³C NMR (CDCl₃, 75 MHz): δ 157.2 (d, J=239 Hz), 148.1, 117.8 (d, J=8 Hz)115.5 (d, J=22 Hz), 52.9, 52.4, 50.2, 12.1.

Example 21

1-ethyl-4-(3-fluorophenyl)piperazine (1s) was obtained by reaction of3-fluoroiodobenzene (2.33 g, 1.23 mL, 10.50 mmol, 1 equiv) and1-ethylpiperazine (2.0 mL, 15.75 mmol, 1.5 equiv) (yellow oil; 697 mg,75% yield); R_(f)=0.49 in CH₂Cl₂/MeOH 9/1; ¹H NMR (CDCl₃, 300 MHz): δ7.23-7.12 (m, 1H), 6.71-6.64 (m, 1H), 6.63-6.55 (m, 1H), 6.55-6.47 (m,1H), 3.27-3.17 (m, 4H), 2.64-2.54 (m, 4H), 2.47 (q, J=7.2 Hz, 2H), 1.13(t, J=7.2 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 164.0 (d, J=243 Hz), 153.1(d, J=10 Hz), 130.2 (d, J=10 Hz), 111.1 (d, J=2 Hz), 105.8 (d, J=22 Hz),102.7 (d, J=25 Hz), 52.8, 52.5, 48.7, 12.1.

Example 22

General Procedure B for ethylation to quaternary ammonium salts 2a-2s:In a sealed vial, the coupled compound (1 equiv) was dissolved in dryTHF; after addition of some copper as stabilizer, iodoethane (7 equiv)was added, and the resulting mixture was heated at 90° C. until completeconsumption of the starting material (TLC in methylene chloride/methanol9/1). Upon completion, the mixture was cooled to room temperature andthe solvent removed in vacuo. The crude was then purified bycrystallization, directly or after a chromatography (silica column,elution in methylene chloride/methanol 9:1. Except as noted, allquaternary ammonium derivatives were crystallized from a mixture of hotTHF and ethanol, with addition of a few drops of hexanes.

Example 23

1,1-diethyl-4-p-tolylpiperazin-1-ium iodide (2a). Obtained from1-ethyl-4-ptolylpiperazine, 1a (50 mg, 0.25 mmol, 1 equiv) and Etl (141μL), followed by crystallization in 2-propanol. Yellow solid (60 mg, 68%yield); m.p.=147.4-148.8° C.; R_(f)=0.37 in CH₂Cl₂/MeOH 8/2; ¹H NMR(DMSO-d6, 300 MHz): δ 7.09 (d, J=8.3 Hz, 2H), 6.91 (d, J=8.4 Hz, 2H),3.58-3.50 (m, 4H), 3.51-3.37 (m, 8H), 2.22 (s, 3H), 1.21 (t, J=7.2 Hz,6H). ¹³C NMR (DMSO-d6, 75 MHz): δ 147.2, 129.5, 128.8, 115.8, 56.5,51.9, 42.0, 20.1, 6.7. HMRS [M]⁺ (C₁₅H₂₅N₂ ⁺) Calcd 233.2012, Found233.2017.

Example 24

4-(4-cyanophenyl)-1,1-diethylpiperazin-1-ium iodide (2b). Obtained from4-(4-ethylpiperazin-1-yl)benzonitrile (142 mg, 0.66 mmol, 1 equiv) andEtl (371 μL), followed by silica column and crystallization. Lightyellow crystals (35 mg, 14% yield); m.p.=197.4-198.2° C.; R_(f)=0.31 inCH₂Cl₂/MeOH 8/2; ¹H NMR (CD₃OD, 300 MHz): δ 7.59 (d, J=8.9 Hz, 1H), 7.13(d, J=8.9 Hz, 2H), 3.80-3.71 (m, 4H), 3.71-3.65 (m, 4H), 3.60 (q, J=7.3Hz, 4H), 1.38 (t, J=7.3 Hz, 6H). ¹³C NMR (CD₃OD, 300 MHz): δ 153.6,134.6, 120.6, 116.2, 102.5, 58.2, 54.3, 42.1, 7.6. HMRS [M]⁺ (C₁₅H₂₂N₃⁺) Calcd 244.1808, Found 244.1813.

Example 25

1,1-diethyl-4-(4-methoxyphenyl)piperazin-1-ium iodide (2c). Obtainedfrom 1-ethyl-4-(4-methoxyphenyl)piperazine 1c, (270 mg, 1.23 mmol, 1equiv) and Etl (692 μL), followed by crystallization. Bright yellowishcrystal (334 mg, 72% yield); m.p.=150.5-153.1° C.; R_(f)=0.35 inCH₂Cl₂/MeOH 8/2; ¹H NMR (CD₃OD, 500 MHz): δ 7.01 (d, J=9.1 Hz, 2H), 6.88(d, J=9.1 Hz, 2H), 3.75 (s, 3H), 3.65-3.59 (m, 4H), 3.54 (q, J=7.3 Hz,4H), 3.45-3.39 (m, 4H), 1.36 (t, J=7.3 Hz, 6H). ¹³C NMR (CD₃OD, 126MHz): δ 156.4, 145.0, 119.9, 115.6, 59.0, 56.0, 54.3, 45.4, 7.4. HMRS[M]⁺ (C₁₅H₂₅N₂O⁺) Calcd 249.1961 Found 249.1972.

Example 26

1,1-diethyl-4-m-tolylpiperazin-1-ium iodide (2d). Obtained from1-ethyl-4-mtolylpiperazine 1d, (101 mg, 0.49 mmol, 1 equiv) and Etl (276μL), followed by crystallization. Yellow crystals (77 mg, 43% yield);m.p.=144.0-145.0° C.; R_(f)=0.37 in CH₂Cl₂/MeOH 8/2; ¹H NMR (CD₃OD, 500MHz): δ 7.16 (t, J=7.9 Hz, 1H), 6.88-6.85 (m, 1H), 6.82 (dd, 1H),6.78-6.74 (m, 1H), 3.64-3.59 (m, 4H), 3.56-3.48 (m, 8H), 2.31 (s, 3H),1.40-1.33 (m, 6H). ¹³C NMR (CD₃OD, 126 MHz): δ 150.9, 140.2, 130.1,123.0, 118.3, 114.7, 58.8, 54.2, 44.1, 21.7, 7.4. HMRS [M]⁺ (C₁₅H₂₅N₂ ⁺)Calcd 233.2012 Found 233.2023.

Example 27

4-(4-chlorophenyl)-1,1-diethylpiperazin-1-ium iodide (2e). Obtained from1-(4-chlorophenyl)-4-ethylpiperazine 1e (146 mg, 0.65 mmol, 1 equiv) andEtl (366 μL), followed by crystallization. Yellowish solid (126 mg, 51%yield); m.p.=151.0-152.1° C.; R_(f)=0.33 in CH₂Cl₂/MeOH 8/2; ¹H NMR(CD₃OD, 500 MHz): δ 7.27 (d, J=9.1 Hz, 2H), 7.01 (d, J=9.0 Hz, 2H),3.65-3.60 (m, 4H), 3.57-3.50 (m, 8H), 1.36 (t, J=7.2 Hz, 6H). ¹³C NMR(CD₃OD, 126 MHz): δ 149.6, 130.1, 127.0, 118.9, 58.6, 54.3, 43.8, 7.4.HMRS [M]⁺ (C₁₄H₂₂ClN₂ ⁺) Calcd 253.1466, Found 253.1476 [M], 255.1449[M+2].

Example 28

4-(3-chlorophenyl)-1,1-diethylpiperazin-1-ium iodide (2f). Obtained from1-(3-chlorophenyl)-4-ethylpiperazine 1f (501 mg, 2.23 mmol, 1 equiv) andEtl (1.25 mL), followed by crystallization. White crystals (412 mg, 48%yield); m.p.=155.0-156.3° C.; R_(f)=0.33 in CH₂Cl₂/MeOH 8/2; ¹H NMR(CD₃OD, 300 MHz): δ 7.26 (d, J=8.4 Hz, 1H), 7.05 (t, J=2.2 Hz, 1H), 6.96(ddd, J=8.4, 2.5, 0.9 Hz, 1H), 6.90 (ddd, J=7.9, 1.9, 0.8 Hz, 1H),3.66-3.60 (m, 4H), 3.55 (q, J=7.2 Hz, 4H), 3.60-3.53 (m, 4H), 1.37 (t,J=7.2 Hz, 6H). ¹³C NMR (CD₃OD, 126 MHz): δ 152.1, 136.1, 131.5, 121.6,117.2, 115.6, 58.5, 54.3, 43.5, 7.4. HMRS [M]⁺ (C₁₄H₂₂ClN₂ ⁺) Calcd253.1466, Found 253.1475 [M], 255.1443 [M+2].

Example 29

1,1-diethyl-4-(3-methoxyphenyl)piperazin-1-ium iodide (2g). Obtainedfrom 1-ethyl-4-(3-methoxyphenyl)piperazine 1g (503 mg, 2.28 mmol, 1equiv) and Etl (1.28 mL), followed by crystallization. White crystals(649 mg, 76% yield); m.p.=146.0-147.0° C.; R_(f)=0.35 in CH₂Cl₂/MeOH8/2; ¹H NMR (CD₃OD, 500 MHz): δ 7.19 (t, J=8.2 Hz, 1H), 6.62 (dt, J=8.2,1.4 Hz, 1H), 6.57 (t, J=2.4 Hz, 1H), 6.51 (dd, J=8.2, 2.3 Hz, 1H), 3.77(s, 3H), 3.64-3.60 (m, 4H), 3.54 (q, J=7.1 Hz, 4H), 3.54-3.50 (m, 4H),1.36 (t, J=7.4 Hz, 6H). 13C NMR (CD₃OD, 126 MHz): δ 162.2, 152.2, 131.1,110.1, 107.3, 104.0, 58.7, 55.8, 54.3, 44.0, 7.4. HMRS [M]⁺ (C₁₅H₂₅N₂O⁺)Calcd 249.1961, Found 249.1966.

Example 30

1,1-diethyl-4-o-tolylpiperazin-1-ium iodide (2h). Obtained from1-ethyl-4-o-tolylpiperazine 1h, (385 mg, 1.88 mmol, 1 equiv) and Etl(1.06 mL), followed by crystallization in THF/ethanol/MeOH. Bright whitecrystals (301 mg, 45% yield); m.p.=158.5-160.2° C.; R_(f)=0.37 inCH₂Cl₂/MeOH 8/2; ¹H NMR (CD₃OD, 500 MHz): δ 7.26-7.16 (m, 3H), 7.07-7.01(m, 1H), 3.67-3.62 (m, 4H), 3.60 (q, J=7.4 Hz, 4H), 3.29-3.23 (m, 4H),2.33 (s, 3H), 1.38 (t, J=7.1 Hz, 6H). ¹³C NMR (CD₃OD, 126 MHz): δ 147.2,129.5, 128.8, 115.8, 56.5, 51.9, 42.0, 20.1, 6.7. HMRS [M]⁺ (C₁₅H₂₅N₂ ⁺)Calcd 233.2012 Found 233.2023.

Example 31

4-(3-cyanophenyl)-1,1-diethylpiperazin-1-ium iodide (2i). Obtained from3-(4-ethylpiperazin-1-yl)benzonitrile 1i, (555 mg, 2.58 mmol, 1 equiv)and Etl (1.45 mL), followed by crystallization from THF/ethanol/MeOH.Colorless crystals (140 mg, 15% yield); m.p.=210-212° C.; R_(f)=0.31 inCH₂Cl₂/MeOH 8/2; ¹H NMR (CD₃OD, 300 MHz): δ 7.50-7.40 (m, 1H), 7.40-7.32(m, 2H), 7.22 (dt, J=7.4, 1.3 Hz, 1H), 3.71-3.62 (m, 8H), 3.58 (q, J=7.3Hz, 4H), 1.38 (t, J=8.2 Hz, 6H). ¹³C NMR (CD₃OD, 75 MHz): δ 151.3,131.5, 124.9, 121.7, 119.9, 119.9, 114.2, 58.4, 54.2, 43.1, 7.4. HMRS[M]⁺ (C₁₅H₂₅N₂ ⁺) Calcd 244.1808 Found 244.1812.

Example 32

4-(2-chlorophenyl)-1,1-diethylpiperazin-1-ium iodide (2j). Obtained from1-(2-chlorophenyl)-4-ethylpiperazine 1j, (110 mg, 0.49 mmol, 1 equiv)and Etl (276 μL), followed by crystallization. White crystals (40 mg,21% yield); m.p.=154.1-156.8° C.; R_(f)=0.33 in CH₂Cl₂/MeOH 8/2; ¹H NMR(CD₃OD, 300 MHz): δ 7.47-7.39 (m, 1H), 7.37-7.28 (m, 2H), 7.17-7.06 (m,1H), 3.72-3.55 (m, 8H), 3.49-3.39 (m, 4H), 1.38 (t, J=7.2 Hz, 5H). ¹³CNMR (CD₃OD, 75 MHz): δ 148.4, 131.7, 130.0, 129.2, 126.5, 122.4, 59.3,54.5, 45.7, 7.4. HMRS [M]⁺ (C₁₄H₂₂ClN₂ ⁺) Calcd 253.1472, Found 253.1483[M], 255.1437 [M+2].

Example 33

1,1-diethyl-4-(3-hydroxyphenyl)piperazin-1-ium iodide (2k). Obtainedfrom 3-(4-ethylpiperazin-1-yl)phenol 1k, (489 mg, 2.37 mmol, 1 equiv)and Etl (1.33 mL), followed by crystallization from ethanol. Off-whitesolid (186 mg, 22% yield); m.p.=200-203° C.; R_(f)=0.38 in CH₂Cl₂/MeOH75/25; ¹H NMR (DMSO-d6, 300 MHz): δ 9.26 (s, 1H), 7.04 (t, J=8.1 Hz,1H), 6.48-6.41 (m, 1H), 6.38-6.34 (m, 1H), 6.33-6.27 (m, 1H), 3.58-3.38(m, 12H), 1.21 (t, J=7.1 Hz, 6H). ¹³C NMR (DMSO-d6, 75 MHz): δ 158.1,150.7, 129.7, 107.1, 106.6, 102.6, 56.4, 51.9, 41.5, 6.8. HMRS [M]⁺(C₁₄H₂₃N₂O⁺) Calcd 235.1805 Found 235.1806.

Example 34

4-(2,4-dimethoxyphenyl)-1,1-diethylpiperazin-1-ium iodide (21). Obtainedfrom 1-(2,4-dimethoxyphenyl)-4-ethylpiperazine 11, (502 mg, 2.01 mmol, 1equiv) and Etl (1.13 mL), followed by crystallization. White solid (236mg, 29% yield); m.p.=187.0-188.8° C.; R_(f)=0.34 in CH₂Cl₂/MeOH 8/2; ¹HNMR (CD₃OD, 300 MHz): δ 7.01 (d, J=8.6 Hz, 1H), 6.57 (d, J=2.7 Hz, 1H),6.49 (dd, J=8.6, 2.7 Hz, 1H), 3.84 (s, 3H), 3.76 (s, 3H), 3.63-3.49 (m,8H), 3.36-3.28 (m, 4H), 1.36 (t, J=7.3 Hz, 6H). ¹³C NMR (CD₃OD, 75 MHz):δ 158.8, 155.2, 133.8, 121.5, 105.3, 100.9, 59.3, 56.2, 56.0, 54.5,45.6, 7.5. HMRS [M]⁺ (C₁₈H₂₇N₂O₂ ⁺) Calcd 279.2067 Found 279.2078.

Example 35

1,1-diethyl-4-(naphthalen-2-yl)piperazin-1-ium iodide (2m). Obtainedfrom 1-ethyl-4-(naphthalen-2-yl)piperazine 1m, (516 mg, 2.15 mmol, 1equiv) and Etl (1.21 mL), followed by crystallization. Yellowish solid(430 mg, 51% yield); m.p.=218-219° C.; R_(f)=0.38 in CH₂Cl₂/MeOH 75/25;¹H NMR (DMSO-d6, 300 MHz): δ 7.86-7.81 (m, 1H), 7.81-7.73 (m, 2H), 7.43(ddd, J=8.1, 5.4, 1.9 Hz, 2H), 7.35-7.26 (m, 2H), 3.66-3.58 (m, 8H),3.52 (q, J=7.1 Hz, 4H), 1.25 (t, J=7.2 Hz, 6H). ¹³C NMR (DMSO-d6, 75MHz): δ 147.1, 134.0, 128.6, 128.1, 127.3, 126.6, 126.4, 123.5, 118.5,109.8, 56.4, 52.0, 41.8, 6.8. HMRS [M]⁺ (C₁₈H₂₅N₂ ⁺) Calcd 269.2018.Found 269.2013.

Example 36

1,1-diethyl-4-(4-(trifluoromethyl)phenyl)piperazin-1-ium iodide (2n).Obtained from 1-ethyl-4-(4-(trifluoromethyl)phenyl)piperazine in, (714mg, 2.76 mmol, 1 equiv) and Etl (1.60 mL), afforded 546 mg (48% crude)of a yellow solid that was crystallized from methanol. White crystals(70 mg, 6% yield); m.p.=175.2-175.5° C. R_(f)=0.48 in CH₂Cl₂/MeOH 75/25;¹H NMR (DMSO-d6, 300 MHz): δ 7.59 (d, J=8.5 Hz, 2H), 7.15 (d, J=8.6 Hz,2H), 3.68-3.59 (m, 4H), 3.59-3.53 (m, 4H), 3.48 (q, J=7.2 Hz, 4H), 1.23(t, J=7.2 Hz, 6H). ¹³C NMR (DMSO-d6, 75 MHz): δ 151.9, 124.8 (q, J=271Hz), 126.2 (q, J=4 Hz), 119.0 (q, J=32 Hz), 114.6, 56.1, 51.9, 40.5,6.8. HMRS [M]⁺ (C₁₅H₂₂F₃N₂ ⁺) Calcd 287.1730. Found 287.1723. Comparisonwith the ¹H NMR spectrum prior to recrystallization revealed the crudeproduct was at least 95% pure.

Example 37

1,1-diethyl-4-(3-(trifluoromethyl)phenyl)piperazin-1-ium iodide (20).Obtained from 1-ethyl-4-(3-(trifluoromethyl)phenyl)piperazine 10, (701mg, 2.71 mmol, 1 equiv) and Etl (1.53 mL), followed by crystallizationfrom methanol. (235 mg, 21% yield); NMR analysis indicated approximately5 mol equivalents of methanol in the crystals, m.p.=207-208° C.;R_(f)=0.48 in CH₂Cl₂/MeOH 75/25; ¹H NMR (CD₃OD, 300 MHz): δ 7.52-7.43(m, 1H), 7.32-7.26 (m, 2H), 7.23-7.16 (m, 1H), 3.70-3.61 (m, 8H), 3.56(q, J=7.3 Hz, 4H), 3.34 (s, methanol), 1.44-1.32 (m, 6H). ¹³C NMR(CD³OD, 75 MHz): 151.3, 132.5 (q, J=32 Hz), 131.2, 125.7 (q, J=273 Hz),120.7, 118.0, 113.5, 58.5, 54.3, 43.4, 7.5. HMRS [M]⁺ (C₁₅H₂₂F₃N₂ ⁺)Calcd 287.1730 Found 287.1738.

Example 38

4-(3-bromophenyl)-1,1-diethylpiperazin-1-ium iodide (2p). Obtained from1-(3-bromophenyl)-4-ethylpiperazine 1p, (768 mg, 2.85 mmol, 1 equiv) andEtl (1.60 mL), followed by crystallization. Brownish white-off crystals(403 mg, 33% yield); m.p.=198.8-199.5° C.; R_(f)=0.37 in CH₂Cl₂/MeOH75/25; ¹H NMR (CD₃OD, 300 MHz): δ 7.23-7.16 (m, 2H), 7.08-6.98 (m, 2H),3.67-3.50 (m, 12H), 1.37 (t, J=7.2 Hz, 6H). ¹³C NMR (CD₃OD, 75 MHz) δ152.3, 131.8, 124.6, 124.1, 120.1, 116.0, 58.5, 54.2, 43.5, 7.5. HMRS[M]⁺ (C₁₄H₂₂BrN₂ ⁺) Calcd 297.0966 Found 297.0963 [M], 299.0944 [M+2].

Example 39

4-(4-bromophenyl)-1,1-diethylpiperazin-1-ium iodide (2q). Obtained from1-(4-bromophenyl)-4-ethylpiperazine 1q, (517 mg, 1.92 mmol, 1 equiv) andEtl (1.08 mL), followed by crystallization. White crystals (220 mg, 27%yield); m.p.=182.5-183.6° C.; R_(f)=0.37 in CH₂Cl₂/MeOH 75/25; ¹H NMR(CD₃OD, 300 MHz): δ 7.40 (d, J=9.0 Hz, 1H), 6.97 (d, J=9.0 Hz, 2H),3.67-3.48 (m, 12H), 1.42-1.32 (m, 6H). ¹³C NMR (CD₃OD, 75 MHz): δ 150.0,133.1, 119.2, 114.1, 58.5, 54.16, 43.7, 7.3. HMRS [M]⁺ (C₁₄H₂₂BrN₂ ⁺)Calcd 297.0961 Found 297.0965 [M], 299.0945 [M+2].

Example 40

1,1-diethyl-4-(4-fluorophenyl)piperazin-1-ium iodide (2r). Obtained from1-ethyl-4-(4-fluorophenyl)piperazine 1r, (704 mg, 3.38 mmol, 1 equiv)and Etl (1.90 mL), followed by crystallization. White crystals (478 mg,39% yield); m.p.=194.0-194.3° C.; R_(f)=0.31 in CH₂Cl₂/MeOH 8/2; ¹H NMR(CD₃OD, 300 MHz): δ 7.08-7.04 (m, 2H), 7.04-7.01 (m, 2H), 3.66-3.59 (m,4H), 3.58-3.50 (m, 4H), 3.50-3.43 (m, 4H), 1.43-1.30 (m, 6H). ¹³C NMR(CD₃OD, 75 MHz): δ 159.2 (d, J=240 Hz), 147.6, 119.7 (d, J=8 Hz), 116.6(d, J=23 Hz), 58.8, 54.3, 44.8, 7.5 HMRS [M]⁺ (C₁₄H₂₂FN₂ ⁺) Calcd237.1762 Found 237.1767.

Example 41

1,1-diethyl-4-(3-fluorophenyl)piperazin-1-ium iodide (2s). Obtained from1-ethyl-4-(3-fluorophenyl)piperazine 1s, (801 mg, 3.85 mmol, 1 equiv)and Etl (2.17 mL), followed by purification via chromatography, crystalswere not obtained. Off-white solid (901 mg, 64% yield),m.p.=125.0-127.0° C.; R_(f)=0.33 in CH₂Cl₂/MeOH 8/2; ¹H NMR (DMSO d6,300 MHz): δ 7.35-7.23 (m, 1H), 6.92-6.80 (m, 2H), 6.71-6.61 (m, 1H),3.62-3.51 (m, 8H), 3.48 (q, J=7.6 Hz, 4H), 1.22 (t, J=7.2 Hz, 6H). ¹³CNMR (DMSO-d6, 75 MHz): δ 163.1 (d, J=241 Hz), 151.1 (d, J=10 Hz), 130.5(d, J=10 Hz), 111.0 (d, J=2 Hz), 105.7 (d, J=21 Hz), 102.1 (d, J=25 Hz),56.1, 51.8, 41.1, 6.8. HMRS [M]⁺ (C₁₄H₂₂FN₂ ⁺) Calcd 237.1762 Found237.1760.

Example 42

General procedure for hydration of nitrile to amide with acetaldoxime:In an oven-dried, sealed vial under argon atmosphere, a mixture ofbenzonitrile derivative (2 equiv), acetaldoxime (4 equiv) andPd(PPh₃)₄(0.1 equiv) in EtOH was heated to reflux until completeconsumption of the starting material (TLC on neutral alumina CH₂Cl₂/MeOH95/5 or 85/15). Upon completion, the reaction mixture was cooled to roomtemperature, filtered through a Celite pad and washed with hot EtOH.After removal of solvent, the crude was purified with neutral aluminachromatography (CH₂Cl₂/MeOH 95/5 to 85/15) and the pooled fractions werethen crystallized from methanol or ethanol.

Example 43

4-(4-carbamoylphenyl)-1,1-diethylpiperazin-1-ium iodide (2t). Obtainedfrom 4-(4-cyanophenyl)-1,1-diethylpiperazin-1-ium iodide 2b (230 mg,0.62 mmol, 1 equiv) and acetaldoxime (73 mg, 1.24 mmol, 2 equiv),followed by crystallization from ethanol. White solids (18 mg, 7%yield); m.p.=238-239° C.; R_(f)=0.42 in CH₂Cl₂/MeOH 8/2 (aluminaneutral); ¹H NMR (CD₃OD, 300 MHz): δ 7.84 (d, J=8.8 Hz, 2H), 7.07 (d,J=8.9 Hz, 2H), 3.71-3.60 (m, 8H), 3.55 (q, J=7.3 Hz, 4H), 1.37 (t, J=7.3Hz, 6H). ¹³C NMR (CD₃OD, 75 MHz): δ 171.9, 153.4, 130.3, 125.8, 115.7,58.3, 54.1, 42.6, 7.3. HMRS [M]⁺ (C₁₅H₂₄N₃O⁺) Calcd 262.1914 Found262.1923.

Example 44

4-(3-carbamoylphenyl)-1,1-diethylpiperazin-1-ium iodide (2u). Obtainedfrom 4-(3-cyanophenyl)-1,1-diethylpiperazin-1-ium iodide 2i (273 mg,0.74 mmol, 1 equiv) and acetaldoxime (87 mg, 1.48 mmol, 2 equiv),followed by crystallization from methanol. Yellow crystals (77 mg, 27%yield) m.p.=212-213° C.; R_(f)=0.42 in CH₂Cl₂/MeOH 8/2 (neutralalumina); ¹H NMR (CD₃OD, 300 MHz): δ 7.54-7.51 (m, 1H), 7.45-7.35 (m,2H), 7.26-7.21 (m, 1H), 3.68-3.59 (m, 8H), 3.56 (q, J=7.2 Hz, 4H), 3.34(s, methanol), 1.37 (t, J=7.3 Hz, 6H). ¹³C NMR (CD₃OD, 75 MHz): δ 172.3,151.0, 135.9, 130.5, 120.9, 120.8, 116.4, 58.6, 54.2, 43.7, 7.4. HMRS[M]⁺ (C₁₅H₂₄N₃O⁺) Calcd 262.1914 Found 262.1905.

Example 45

1-ethyl-N-phenylpiperidin-4-amine (3). Reductive amination with NaBH₃CN.In a flame-dried and argon-flushed round-bottom flask, aniline (1.41 mL,1.44 g, 15.47 mmol, 1 equiv) was dissolved in dry methanol (freshlydistilled from Na), and 1-ethyl-4-piperidone (2.5 mL, 2.36 mg, 18.56mmol, 1.2 equiv) was added, followed by sodium cyanoborohydride (1.17 g,18.56 mmol, 1.2 equiv). The pH was adjusted to 6.0 (pH strips) withglacial acetic acid and the mixture stirred at room temperature. (TLC inCH₂Cl₂/MeOH 95/5). Even after stirring for seven days and heating atreflux, the reaction did not come to completion. Saturated aqueousNaHCO₃ was added to quench the reaction, and methanol was removed byrotary evaporation. The residue was partitioned between ethyl acetateand water, and extracted with EtOAc three times. The combined organiclayers were dried over MgSO₄, and concentrated in vacuo. The residue waschromatographed on silica gel, eluting in CH₂Cl₂/MeOH 93/7 to give thedesired product as an off-white solid (1.88 g, 59%). m.p.=43.0-44.6° C.;R_(f)=0.33 in CH₂Cl₂/MeOH 75/25; ¹H NMR (CD₃OD, 300 MHz): δ 7.13-7.06(m, 2H), 6.68-6.57 (m, 3H), 3.41-3.32 (m, 1H), 3.11-3.00 (m, 2H), 2.59(q, J=7.3 Hz, 2H), 2.39-2.26 (m, 2H), 2.12-2.01 (m, 2H), 1.60-1.45 (m,2H), 1.15 (t, J=7.3 Hz, 3H). ¹³C NMR (CD₃OD, 75 MHz): δ 148.9, 130.1,118.2, 114.8, 53.2, 52.9, 50.6, 32.4, 11.7. HMRS [M+H]⁺ (C₁₃H₂₁N⁺) Calcd205.1699 Found 205.1701.

Example 46

N-(1-ethylpiperidin-4-yl)-2,2,2-trifluoro-N-phenylacetamide (4). In aflame-dried and argon-flushed round-bottom flask, a solution of1-ethyl-N-phenylpiperidin-4-amine (3) (1.53 g, 7.46 mmol, 1 equiv) indry CH₂Cl₂ (75 mL) was cooled to 0° C. Triethylamine (4.20 mL, 3.02 g,29.9 mmol, 4 equiv) was added, followed by addition dropwise of thetrifluoroacetic anhydride (3.20 mL, 4.70 g, 22.4 mmol, 3 equiv). Theresulting mixture was then stirred at 0° C. until completion (5h) (TLCin CH₂Cl₂/MeOH 95/5). Deionized water was then added at 0° C., theorganic fraction washed with saturated brine, dried over MgSO₄, andconcentrated in vacuo. The crude obtained was purified by a silica gelcolumn eluting with CH₂Cl₂/MeOH 95/5 to 9/1, and then the isolatedfraction was further purified on a silica gel column eluting inEtOAc/MeOH 100/0 to 9/1 affording the desired product as a yellow solid,(911 mg, 41%); m.p.=50.2-51.3° C.; R_(f)=0.5 in CH₂Cl₂/MeOH 9/1; ¹H NMR(CDCl₃, 300 MHz): δ 7.45-7.34 (m, 3H), 7.19-7.10 (m, 2H), 4.55 (tt,J=12.2, 4.0 Hz, 1H), 3.03-2.92 (m, 2H), 2.37 (q, J=7.2 Hz, 2H),2.11-1.98 (m, 2H), 1.89-1.78 (m, 2H), 1.55-1.38 (m, 2H), 1.02 (t, J=7.2Hz, 3H); ¹³C NMR (CDCl₃, 75 MHz): δ 156.7 (q, J=35 Hz), 134.8, 130.6,129.4, 128.9, 116.5 (q, J=289 Hz), 55.3, 52.4, 52.1, 29.7, 12.1. HMRS[M+H]⁺ (C₁₅H₂₀F₃N₂O⁺) Calcd 301.1522 Found 301.1531.

Example 47

1,1-diethyl-4-(2,2,2-trifluoro-N-phenylacetamido)piperidinium iodide(5). In a sealed vial,N-(1-ethylpiperidin-4-yl)-2,2,2-trifluoro-N-phenylacetamide (4) (417 mg,1.39 mmol, 1 equiv) was dissolved in dry THF; after addition of copperas a stabilizer, Etl (782 μL, 7 equiv) was added and the resultingmixture was heated at 90° C. until complete consumption of the startingmaterial (22 h, TLC in CH₂Cl₂/MeOH 95/5). Upon completion, the mixturewas cooled to room temperature and the solvent removed in vacuo. Thecrude was then purified by chromatography (silica column, elution inCH₂Cl₂/MeOH 94/6 to 9/1) to afford purified 5 as a yellow solid (520 mg,yield 82%). m.p.=141.0-142.0° C.; R_(f)=0.4 in CH₂Cl—/MeOH 75/25; ¹H NMR(CD₃OD, 300 MHz): δ 7.58-7.46 (m, 3H), 7.46-7.33 (m, 2H), 4.69-4.53 (m,1H), 3.72-3.59 (m, 2H), 3.53-3.21 (m, 6H), 2.25-1.97 (m, 4H), 1.32 (t,J=6.4 Hz, 3H), 1.22 (t, J=7.4 Hz, 3H); ¹³C NMR (DMSO-d6, 75 MHz): δ155.4 (q, J=34 Hz), 134.9, 130.0, 129.8, 129.2, 115.9 (q, J=289 Hz),57.4, 56.0, 54.9 (CH₂Cl₂), 53.2, 47.3, 22.7, 7.1, 6.7. HMRS [M]⁺(C₁₇H₂₄F₃N₂O⁺) Calcd 329.1835 Found 329.1849.

Example 48

1,1-diethyl-4-(phenylamino)piperidinium iodide (6). In a round-bottomflask flushed with argon,1,1-diethyl-4-(2,2,2-trifluoro-N-phenylacetamido)piperidinium iodide (5)(400 mg, 0.88 mmol, 1 equiv) was dissolved in MeOH/water 7/1 (49/7 mL);K₂CO₃ (1.46 g, 10.56 mmol, 12 equiv) was added at room temperature andthe resulting mixture was then heated at 65° C. until completion (1h,TLC CH₂Cl₂/MeOH 9/1). The mixture was then cooled to room temperatureand the solvents were removed under vacuum. The crude was dissolved inMeOH and filtered through cotton to remove precipitates, and thefiltered solution was concentrated in vacuo.

Purification on a silica column (CH₂Cl₂/MeOH 9/1 to 85/15) afforded thepure compound as a light yellow solid (173 mg), which was thencrystalized from hot ethanol to obtain white crystals, (79 mg, 25%);m.p.=203-204° C.; R_(f)=0.37 in CH₂Cl₂/MeOH 75/25; ¹H NMR (CD₃OD, 500MHz): δ 7.12 (t, J=7.5 Hz, 2H), 6.71 (d, J=7.9 Hz, 2H), 6.65 (t, J=7.4Hz, 1H), 3.76-3.68 (m, 1H), 3.63-3.56 (m, 2H), 3.50 (q, J=7.1 Hz, 2H),3.46-3.36 (m, 4H), 2.25-2.15 (m, 2H), 1.94-1.83 (m, 2H), 1.35 (t, J=7.3Hz, 3H), 1.31 (t, J=8.0 Hz, 3H); ¹³C NMR (DMSO-d6, 75 MHz): δ 147.2,129.1, 116.3, 112.8, 55.6, 55.0, 49.5, 45.4, 25.0, 7.2, 6.7. HMRS [M]⁺(C₁₅H₂₅N₂ ⁺) Calcd 233.2012 Found 233.2010.

Example 49

4-benzylidene-1-ethylpiperidine (7). In a flame-dried and argon-flushedroundbottom flask, to a solution of diethyl benzylphosphonate (1.2 mL,1.27 g, 5.57 mmol, 1.5 equiv) and 15-crown-5 (150 μL, 167 mg, 0.76 mmol,0.2 equiv) in dry THF (17 mL) at 0° C. was added sodium hydride 60%dispersion in mineral oil (223 mg, 5.57 mmol, 1.5 equiv, portion-wiseaddition). After stirring 40 min at 0° C., a solution of1-ethyl-4-piperidone (500 μL, 472 mg, 3.71 mmol, 1 equiv) in dry THF (30mL) was added dropwise at 0° C. After stirring at 0° C. for 10 min, thereaction mixture was allowed to warm up and stir at room temperatureuntil completion (TLC in CH₂Cl₂/MeOH 9/1), which occurred after 4 days.The mixture was cooled to 0° C., diluted with deionized water, and thenthe product was extracted three times with EtOAc. The organic layer waswashed with saturated aqueous NaHCO₃ and saturated brine, then driedover MgSO₄. The solvent was removed under reduced pressure to yield ayellow oil that was purified on a silica gel column (CH₂Cl₂/MeOH 96/4)to afford the desired product as a pale yellowish oil (337 mg, 45%yield). R_(f)=0.33 in CH₂Cl₂/MeOH 85/15; ¹H NMR (CD₃OD, 300 MHz): δ7.33-7.26 (m, 2H), 7.21-7.15 (m, 3H), 6.35 (s, 1H), 2.65 (dd, J=6.5, 5.1Hz, 2H), 2.58-2.49 (m, 6H), 2.48-2.42 (m, 2H), 1.15 (t, J=7.3 Hz, 3H);¹³C NMR (CD₃OD, 75 MHz): δ 139.2, 138.8, 129.9, 129.2, 127.4, 125.1,55.7, 54.9, 53.2, 36.4, 29.3, 11.8.

Example 50

4-benzylidene-1,1-diethylpiperidinium iodide (8). In a sealed vial,4-benzylidene-1-ethylpiperidine (7) (337 mg, 1.67 mmol, 1 equiv) wasdissolved in EtOH; after addition of some copper metal as stabilizer,Etl (940 μL, 7 equiv) was added and the resulting mixture was heated at90° C. until complete consumption of the starting material (TLC inCH₂Cl₂/MeOH 9/1). Upon completion (51 h), the mixture was cooled to roomtemperature and the solvent removed in vacuo. The crude was thenpurified by a silica gel column eluting with CH₂Cl₂/MeOH 95/5 to 75/25;the product was then further purified by a second silica column usingthe same eluent and by crystallization in ethanol/methanol 1:1,affording 18 mg of pure product (3%) as white crystals. m.p.=84.5-86.0°C.; R_(f)=0.34 in CH₂Cl₂/MeOH 8/2; ¹H NMR (CD₃OD, 300 MHz): δ 7.41-7.30(m, 2H), 7.30-7.20 (m, 3H), 6.59 (s, 1H), 3.51 (q, J=7.1 Hz, 6H),3.41-3.34 (m, 2H), 2.91-2.81 (m, 2H), 2.81-2.73 (m, 2H), 1.34 (tt,J=7.3, 1.9 Hz, 6H); ¹³C NMR (CD₃OD, 126 MHz): δ 137.6, 132.2, 129.9,129.5, 128.7, 128.2, 59.9, 59.2, 54.1, 30.3, 24.1, 7.5. HMRS [M]⁺(C₁₈H₂₄N⁺) Calcd 230.1903 Found 230.1900.

Example 50

TABLE 3 Compound R₁ R₂ R₃ 1a 2a CH₃ H H 1b 2b CN H H 1c 2c OCH₃ H H 1d2d H CH₃ H 1e 2e Cl H H 1f 2f H Cl H 1g 2g H OCH₃ H 1h 2h H H CH₃ 1i 2iH CN H 1j 2j H H Cl 1k 2k H OH H 1l 2l OCH₃ H OCH₃ 1n 2n CF₃ H H 1o 2o HCF₃ H 1p 2p H Br H 1q 2q Br H H 1r 2r F H H 1s 2s H F H 2t CONH₂ H H 2uH CONH₂ H

Example 51

Electrophysiology: New compounds were assayed for activity with the α7nAChR expressed in Xenopus oocytes using two-electrode voltage clampingas previously described in Papke et al., (2014) J. Pharmacol.Experimental Therap. 350: 665-680, incorporated herein by reference inits entirety, and compared to responses evoked by 60 μM Ach (FIGS. 5 and6). The compound set was assayed at a concentration of 30 μM to providea standard comparison benchmark.

Based on earlier experience this concentration is high enough to providean observable response and low enough to avoid possible complicationssuch as channel block. The net-charge response for each compoundapplication is reported relative to those for ACh control applications.Data were expressed as the mean±S.E.M. from at least four oocytes foreach experiment and were plotted by Kaleidagraph (Abelbeck Software,Reading, Pa.).

TABLE 1 Compounds tested in this study. Relative Potentiated Compoundresponse^(a) response^(a) diEPP 0.002 ± 0.003  1.3 ± 0.3 1f, m-chloroPEP 0.009 ± 0.008  0.1 ± 0.1 1n, p-CF3 PEP 0.000 ± 0.004 1.4 ± 0. 1p,m-bromo PEP 0.000 ± 0.003  6.9 ± 2.4 1r, p-fluoro PEP 0.000 ± 0.003  0.0± 0.0 2a, p-methyl 0.011 ± 0.003  3.6 ± 2.0 2b, p-cyano 0.234 ± 0.024 68.0 ± 25.4 2c, p-methoxy 0.063 ± 0.014 11.6 ± 0.3 2d, m-methyl 0.001 ±0.000  0.2 ± 0.1 2e, p-chloro 0.234 ± 0.048 18.3 ± 6.2 2f, m-chloro0.020 ± 0.003  4.2 ± 1.0 2g, m-methoxy 0.011 ± 0.011  0.6 ± 0.2 2h,o-methyl 0.319 ± 0.066 16.4 ± 2.4 2i, m-cyano 0.022 ± 0.024  1.5 ± 0.72j, o-chloro 0.367 ± 0.054 36.6 ± 6.1 2k, m-OH 0.283 ± 0.013 31.4 ± 5.12l, p,o-dimethoxy 0.014 ± 0.009  1.1 ± 0.1 2m, 2-naphthalene 0.143 ±0.018 15.9 ± 2.3 2n, p-CF₃ 0.032 ± 0.003 61.8 ± 7.7 2o, m-CF₃ 0.000 ±0.002  3.1 ± 2.1 2p, m-bromo 0.040 ± 0.006 10.0 ± 1.7 2q, p-bromo 0.270± 0.017 12.3 ± 3.5 2r, p-fluoro 0.010 ± 0.002 22.9 ± 5.8 2s, m-fluoro0.061 ± 0.089  0.2 ± 0.1 2t, p-CONH₂ 0.065 ± 0.009 43.9 ± 8.5 2u,m-CONH₂ 0.056 ± 0.022 50.7 ± 8.5 6, diEPP analogue 0.002 ± 0.004  5.6 ±2.1 8, diEPP analogue 0.071 ± 0.027  9.9 ± 3.1 ^(a)Values are the mean ±SEM for N ≥ 4 experiments. All data are relative to the response of thereceptor to 60 μM control applications of ACh. “Relative response”refers to receptor response to a 30 μM application of test compound.Potentiated response refers to receptor response with 30 μM testcompound and 10 μM PNU-120596 co-application.

TABLE 2 Partial agonist properties Estimated Rank Peak PNU Compoundcurrent^(a) Net Charge^(a) Ratio potency efficacy Response^(a)o-chloro(2j) 0.23 ± 0.03 0.37 ± 0.05 0.63 4 2 37 ± 6  o-methyl(2h) 0.15± 0.02 0.32 ± 0.07 0.46 5 1 16 ± 2  m-hydroxy(2k) 0.19 ± 0.02 0.28 ±0.02 0.67 3 4 31 ± 5  naphthalene(2m) 0.10 ± 0.02 0.14 ± 0.02 0.71 1 616 ± 2  p-chloro(2e) 0.17 ± 0.02 0.23 ± 0.05 0.71 1 5 18 ± 6 p-cyano(2b) 0.10 ± 0.01 0.23 ± 0.02 0.43 6 3 68 ± 25 ^(a)Relative to 60μM ACh controls (n ≥ 4)

Example 52

Synthesis of para-pentafluorosulfanyl diEPP (bromine salt) (5.MQ.65):The present compound was synthesized according to the procedure as shownin FIG. 1. 1-ethyl-piperazine (1.5 equiv) was reacted with4-bromophenylsulfur pentafluoride (1 equiv), K₃PO₄ (2 equiv), CuI (0.1equiv) and L-proline (0.2 equiv) in DMSO at 90-100° C. for 17 h to give5.MQ.65. (b) EtBr 10 equiv., dry THF, 80-90° C., 118 h to give 5.MQ.67.

Example 53

Synthesis of cyclohexil diEPP (2.MQ.173.2): 1-phenylpiperazinehydrochloride was dissolved in NaOH 1M and the aqueous phase wasextracted five times with diethyl ether to give, after evaporation ofthe solvent, 1-phenylpiperazine as free base. 1-phenylpiperazine wasdissolved in CH₃CN, K₂CO₃ (1.5 equiv) was added, followed by1,5-diiodopentane (1 equiv). The reaction mixture was heated and stirredat 75° C. for 2 h. After evaporation of the solvent, the crude mixturewas purified over a silica gel column chromatography, eluting inCH₂Cl₂/MeOH 95:5 to 8:2. The isolated product was then re-crystalizedfrom a mixture of THF/EtOH/MeOH to give 2.MQ.173.2 having the formula:

1-20. (canceled)
 21. A compound having the formula III, or IV:

or a pharmaceutically acceptable salt thereof, wherein: Y is:

Z is

n is 2, 3, 4, or 5; R₁, R₂, and R₃ is each independently a hydrogen, analkyl group, cyano, an alkoxy group, a halogen, a trihaloalkyl, acarboxamide, pentafluorosulfanyl, or hydroxyl, and wherein the halogenis fluorine, chlorine or bromine; and R₅ is a hydrogen or acarboxytrifluoromethyl.
 22. The compound of claim 21, wherein R₁, R₂,and R₃ is each independently a hydrogen, a methyl, cyano, methoxy, ahalogen, trihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl.23. The compound of claim 21, wherein R₁, R₂, and R₃ is eachindependently a hydrogen, a methyl, cyano, methoxy, a halogen, atrihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl, and R₅ ishydrogen.
 24. The compound of claim 21, wherein: when R₁ is a methyl ora CN, R₂ and R₃ are each hydrogen; when R₁ is a methoxy, R₂ is hydrogenR₃ is hydrogen or methoxy; when R₁ is a halogen, R₂ and R₃ are eachhydrogen; when R₁ is a trifluoromethyl, R₂ and R₃ are each hydrogen;when R₁ is a carboxamide, pentafluorosulfanyl, R₂ and R₃ are eachhydrogen; when R₁ and R₃ are each hydrogen, R₂ is a methyl, a CN, amethoxy, a halogen, a trifluoromethyl, or a carboxamide, or OH; and whenR₁ and R₂ are each hydrogen, R₃ is a methyl or Cl.
 25. A pharmaceuticalcomposition comprising a compound having the formula III, or IV:

or a pharmaceutically acceptable salt thereof, wherein: Y is:

Z is

n is 2, 3, 4, or 5; R₁, R₂, and R₃ is each independently a hydrogen, analkyl group, cyano, an alkoxy group, a halogen, a trihaloalkyl, acarboxamide, pentafluorosulfanyl, or hydroxyl, and wherein the halogenis fluorine, chlorine or bromine; R₄ is a hydrogen or an ethyl group;and R₅ is a hydrogen or a carboxytrifluoromethyl; and a pharmaceuticallyacceptable carrier.
 26. The pharmaceutical composition of claim 25,wherein R₁, R₂, and R₃ is each independently a hydrogen, a methyl,cyano, methoxy, a halogen, trihaloalkyl, a carboxamide or hydroxyl. 27.The pharmaceutical composition of claim 25, wherein R₁, R₂, and R₃ iseach independently a hydrogen, a methyl, cyano, methoxy, a halogen, atrihaloalkyl, a carboxamide, pentafluorosulfanyl, or hydroxyl, and R₅ ishydrogen.
 28. The pharmaceutical composition of claim 25, wherein: whenR₁ is a methyl or a CN, R₂ and R₃ are each hydrogen; when R₁ is amethoxy, R₂ is hydrogen R₃ is hydrogen or methoxy; when R₁ is a halogen,R₂ and R₃ are each hydrogen; when R₁ is a trifluoromethyl, R₂ and R₃ areeach hydrogen; when R₁ is a carboxamide, pentafluorosulfanyl, R₂ and R₃are each hydrogen; when R₁ and R₃ are each hydrogen, R₂ is a methyl, aCN, a methoxy, a halogen, a trifluoromethyl, or a carboxamide, or OH;and when R₁ and R₂ are each hydrogen, R₃ is a methyl or Cl.
 29. Thepharmaceutical composition of claim 6, wherein said composition isformulated to deliver to a human or animal subject in need thereof, anamount of the compound therapeutically effective in modulating theactivity of a nicotinic acetylcholine receptor in the recipient patient,and wherein the therapeutically effective amount is delivered as asingle dose or as a series of doses.
 30. The pharmaceutical compositionof claim 25, further comprising a therapeutically effective amount of anicotinic acetylcholine receptor positive allosteric modulator (PAM).31. The composition of claim 30, wherein the nicotinic acetylcholinereceptor positive allosteric modulator (PAM) is a type II PAM.
 32. Thecomposition of claim 31, wherein the nicotinic acetylcholine receptorpositive allosteric modulator (PAM) is the type II PAM1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea(PNU-120596).
 33. A method of modulating the activity of a nicotinicacetylcholine receptor in an animal or human subject by administering tosaid subject therapeutically effective doses of a silent agonist of thenicotinic acetylcholine receptor and a nicotinic acetylcholine receptorpositive allosteric modulator (PAM).
 34. The method of claim 33, whereinthe silent agonist of the nicotinic acetylcholine receptor and thenicotinic acetylcholine receptor positive allosteric modulator (PAM) areadministered to the subject simultaneously or as consecutive doses. 35.The method of claim 33, wherein the silent agonist of the nicotinicacetylcholine receptor is a compound having the formula I, II, III, orIV:

or a pharmaceutically acceptable salt thereof, wherein: Y is:

Z is

n is 2, 3, 4, or 5; R₁, R₂, and R₃ is each independently a hydrogen, analkyl group, cyano, an alkoxy group, a halogen, a trihaloalkyl, acarboxamide, pentafluorosulfanyl, or hydroxyl, and wherein the halogenis fluorine, chlorine or bromine; and R₅ is a hydrogen or acarboxytrifluoromethyl.
 36. The method of claim 15, wherein thenicotinic acetylcholine receptor positive allosteric modulator (PAM) isa type II PAM.
 37. The method of claim 18, wherein the nicotinicacetylcholine receptor positive allosteric modulator (PAM) is the typeII PAM 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea(PNU-120596).